Probiotic compositions for treating and preventing colorectal cancer

ABSTRACT

The present invention provides for compositions and methods for treating colorectal cancer or for reducing the risk of developing the disease in an individual.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 63/229,933, filed Aug. 5, 2021, the contents of which are herebyincorporated by reference in the entirety for all purposes.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing file, entitled,“80015-1342522-033510US_ST26.xml”, was created on Feb. 11, 2023 and is4,194 bytes in size. The information in electronic format of theSequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Colorectal cancer (CRC) is the third most common cancer and the thirdleading cause of cancer mortality worldwide, with an estimated incidenceof 1 million new cases and a mortality of >500 000 deaths per year.Several intrinsic (e.g., age, male gender, ethnicity, diabetes mellitus,obesity and inflammatory bowel disease) and extrinsic (e.g., cigarettesmoking, inadequate intake of fiber, high consumption of alcohol, redmeat and high-fat diet) factors are associated with increased risks forCRC. The epidemiology of CRC is under dynamic changes owing to thechanging prevalence and distribution of risk factors. In this regard,CRC incidence in many developing countries, including Asian countries,has increased 2- to 4-fold over the last two decades and has now reachedan alarming rate, with Westernization of diet playing a pivotal role.

It has been verified about 38 trillion bacteria exist the in humanintestine. Because of their symbiotic and co-operative relationship withthe human body, these bacteria have a close association with thepathogenesis and progression of CRC. The association of CRC with alteredgut microbiota has been studied in different populations, identifyingcertain bacterial species for their potential roles, either beneficialor detrimental, in tumorigenesis.

Several treatment modalities including surgery, chemotherapy, radiationtherapy and targeted therapy (e.g., cetuximab and bevacizumab) have beendevised to manage CRC. However, the prognosis of patients withmetastatic CRC remains dismal, highlighting the importance of preventionas well as early treatment of this disease. The long, stepwiseprogression of CRC from cellular transformation to full-blown metastaticlesions has enabled its prevention through natural compounds or drugs toblock or reverse the process. In particular, economic analysis suggeststhat chemoprevention could be a cost-effective intervention whentargeted at intermediate-risk populations following polypectomy. To thisend, non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2(COX-2) inhibitors have been shown to reduce the occurrence of CRC orits precancerous lesions in high-risk individuals. However, thelong-term use of these agents has been associated with an increased riskof cardiovascular events, posing the concern of high-risk benefit ratiofor recommending these agents for CRC chemoprevention. Thechemo-preventive effects of other agents including folic acid, calcium,vitamin D, and antioxidants have also been explored but their efficaciesremain to be fully established. Probiotics are commensal livingmicroorganisms in the human gut. Managing CRC risk and disease outcomeby way of modifying the profile of gut microorganisms is a highlydesirable means of medical intervention for its high efficacy, low cost,and low risk of side effects. Thus, there exists a pressing need fordeveloping new and effective methods and compositions useful for thepurposes of preventing and treating CRC. This invention fulfills thisand other related needs.

BRIEF SUMMARY OF THE INVENTION

The present inventors discovered in their studies that certain gutmicrobial species and their metabolites are effective for the preventionand treatment of CRC. The microorganisms and metabolites so identifiednow serve to provide new methods and compositions for reducing anindividual's risk of developing CRC at a later time and/or for treatingCRC in an individual who has already been diagnosed with the disease.

In a first aspect, the present invention provides a composition that isuseful for preventing or treating CRC in a human subject, for example, aperson who does not have CRC but is at increased risk for CRC due tofamily history or with a personal medical history of having had coloncysts or polyps in the past, or a person who has already been diagnosedwith CRC. The composition comprises an effective amount of (1) livebacteria Lactobacillus gallinarum or Lactococcus lactis, indole-3-lacticacid (ILA), or an aminopeptidase secreted by L. lactis with a molecularweight of more than 100 kDa, or any combination of two or more of theabove; and (2) one or more physiologically acceptable excipients orcarriers. In some cases, the composition further comprises an effectiveamount of live bacteria Carnobacterium maltaromaticum in addition to L.gallinarum, L. lactis, ILA, or the aminopeptidase produced by L. lactishaving a greater-than 100 kDa molecular weight. In some embodiments, thecomposition comprises (a) L. gallinarum and/or L. lactis and (b) C.maltaromaticum in a combined effective amount. In some embodiments, thecomposition is formulated for oral ingestion, for example, in the formof a food or beverage item, or as an additive to food or beverage. Insome embodiments, the composition is in the form of a powder, liquid,paste, cream, tablet, capsule, or caplet. In some embodiments, thecomposition contains L. gallinarum or L. lactis in the range of about1×10⁸ to about 1×10¹² colony-forming units (CFU) per gram weight of thecomposition. In some embodiments, the composition contains live bacteriaC. maltaromaticum in range of about 1×10⁸ to about 1×10¹² CFU per gramweight of the composition. In some embodiments, the composition containsL. gallinarum (or L. lactis) to C. maltaromaticum at a CFU ratio betweenany two bacterial species among the three bacterial species ranging fromabout 1:5 to about 5:1, for example, from about 1:3 to about 3:1, orfrom about 1:2 to about 2:1, or about 1:1. In some embodiments, thecomposition is formulated in multiple packages each in a daily dosage.

In the second aspect, the present invention provides a method fortreating or preventing CRC in a subject by administering to the subjectan effective amount of the composition described above and herein,namely containing an effective amount of (1) Lactobacillus gallinarum,Lactococcus lactis, ILA, or an aminopeptidase secreted by L. lactis witha molecular weight of greater than 100 kDa, or any combination of two ormore of the above; and (2) one or more physiologically acceptableexcipients. In some embodiments, the subject has been diagnosed withCRC. In other embodiments, the subject has not been diagnosed with CRC.In some cases, the composition comprises an effective amount of (i) livebacteria Lactobacillus gallinarum or L. lactis; (ii) L. gallinarum or L.lactis culture supernatant; (iii) ILA or an aminopeptidase that isproduced by L. lactis and has a molecular weight higher than 100 kDa; or(iv) any combination of two or more of (i), (ii), and (iii). In someembodiments, the composition further comprises an effective amount oflive bacteria Carnobacterium maltaromaticum. In some embodiments, themethod includes as the administering step administration to the subjecta first composition comprising an effective amount of (i) live bacteriaL. gallinarum or L. lactis; (ii) L. gallinarum or L. lactis culturesupernatant; (iii) ILA or an aminopeptidase secreted by L. lactis with amolecular weight higher than 100 kDa; or (iv) any combination of two ormore of (i), (ii), and (iii), and administering to the subject a secondcomposition comprising an effective amount of live bacteria C.maltaromaticum. In some cases, the L. gallinarum culture supernatant isthe fraction of <3 kDa in molecular weight. In some cases, the L. lactisculture supernatant is the fraction of >100 kDa in molecular weight. Insome embodiments, the composition used in the method contains livebacteria L. gallinarum (or L. lactis) and C. maltaromaticum each in therange of about 1×10⁸ to about 1×10¹² CFU per gram weight of thecomposition and at a CFU ratio between any two bacterial species amongthe three bacterial species ranging from about 1:5 to about 5:1, forexample, from about 1:3 to about 3:1, or from about 1:2 to about 2:1, orabout 1:1. In some embodiments, one single composition comprising (1)ILA or L. gallinarum, L. lactis, ILA, or an aminopeptidase secreted byL. lactis with a higher than 100 kDa molecular weight and (2) C.maltaromaticum is administered. In some embodiments, two or moreseparate compositions each comprising one or more of live bacteria L.gallinarum or L. lactis, ILA, an aminopeptidase produced by L. lactiswith a molecular weight higher than 100 kDa, and C. maltaromaticum areadministered. In some embodiments, the administration step comprisesoral ingestion of the composition(s). In some embodiments, the subjectin the claimed method is not diagnosed with any of the diseases orconditions previously known to require administration of L. gallinarum,L. lactis, ILA, or C. maltaromaticum. One exemplary of suchdisease/condition is colitis.

In a related aspect, the present invention provides a novel use of acomposition for treating or preventing CRC in a subject as well as amethod of making such a composition. The composition containing aneffective amount of (1) Lactobacillus gallinarum, Lactococcus lactis,ILA, or an L. lactis-produced aminopeptidase with a higher than 100 kDamolecular weight, or any combination of two or more of the above; and(2) one or more physiologically acceptable excipients. Optionally, thecomposition further comprises an effective amount of live bacteriaCarnobacterium maltaromaticum. The composition may serve as a medicamentor food/beverage supplement for the purpose of treating or preventingCRC in a subject with the disease or in a subject without the diseasebut at risk of later developing the disease. The composition may beproduced by combining an effective amount of any one or more of (i) livebacteria Lactobacillus gallinarum or L. lactis; (ii) L. gallinarum or L.lactis culture supernatant; (iii) ILA; or (iv) an L. lactis—producedaminopeptidase having a molecular weight higher than 100 kDa with atleast one possibly more physiologically or pharmaceutically acceptableexcipients or carriers to form a mixture, which may take any one of avariety of forms from liquid, semi-liquid, to solid or powder. In somecases, the L. gallinarum culture supernatant is the <3 kDa molecularweight fraction of the culture broth. In some cases, the culturesupernatant is the >100 kDa molecular weight fraction of the L. lactisculture broth. Optionally, an appropriate amount of live bacteria C.maltaromaticum is further combined in the mixture, preferably toconstitute an effective amount in combination with L. gallinarum, L.lactis, ILA, or an L. lactis-produced aminopeptidase with a >100 kDamolecular weight. In some embodiments, the composition contains livebacteria L. gallinarum (or L. lactis) and C. maltaromaticum each in therange of about 1×10⁸ to about 1×10¹² CFU per gram weight of thecomposition and at a CFU ratio among any two of the three bacterialspecies ranging from about 1:5 to about 5:1, for example, from about 1:3to about 3:1, or from about 1:2 to about 2:1, or about 1:1. In somecases, the composition is formulated for oral ingestion, for example,having been incorporated in a food or beverage item, or as an additiveto food or beverage. In some embodiments, the composition is in the formof a powder, liquid, paste, cream, tablet, capsule, or caplet.

In a third aspect, the present invention provides a kit for treating orpreventing CRC in a subject comprising (1) a first container containinga first composition comprising an effective amount of L. gallinarum, L.lactis, ILA, or an L. lactis—produced aminopeptidase with a >100 kDamolecular weight; and (2) a second container containing a secondcomposition comprising an effective amount of C. maltaromaticum. In someembodiments, the kit includes a first composition containing livebacteria L. gallinarum or L. lactis present in the range of about 1×10⁸to about 1×10¹² CFU per gram weight of the first composition, and asecond composition containing live bacteria C. maltaromaticum is presentin the range of about 1×10⁸ to about 1×10¹² CFU per gram weight of thesecond composition. In some cases, the live bacteria L. gallinarum (orL. lactis) and C. maltaromaticum are present in the first and secondpositions to be administered together at a CFU ratio between any two ofthe three bacterial species ranging from about 1:5 to about 5:1, forexample, from about 1:3 to about 3:1, or from about 1:2 to about 2:1, orabout 1:1. In some embodiments, the compositions are in the form of apowder, liquid, paste, cream, tablet, capsule, or caplet. The kitstypically further include an instruction manual providing directions forthe user to administer the composition(s) to an appropriate recipient(e.g., one who is at risk of CRC but does not have colitis) inaccordance with a pre-determined dosage and frequency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that C. maltaromaticum is depleted in stool samples ofpatients with CRC and predicts overall survival. (A) The abundance of C.maltaromaticum in clinical stool samples from normal controls (n=39) andpatients with CRC (n=39). (B) The relationship between the abundance ofC. maltaromaticum and the survival of CRC patients. Results arepresented as mean±S.D. Each spot represents one subject. Statisticalsignificance was assessed by Student's t-test.

FIG. 2 shows that C. maltaromaticum inhibits the viability and inducesG2/M-phase cell cycle arrest of colon cancer cells. (A) The viability ofCRC cells (HCT116, DLD1) and normal colon cells (NCM460) afterco-incubation with or without C. maltaromaticum. E. coli was used asbacteria control. (B) The cell cycle distribution of colon cells afterco-incubation with or without C. maltaromaticum. E. coli was used asbacteria control. Results are presented as mean±S.D. Statisticalsignificance was determined by one-way ANOVA or two-way ANOVA whereappropriate.

FIG. 3 shows that C. maltaromaticum protects against intestinaltumorigenesis in Apc^(min/+) mice. (A) Schematic diagram showing theexperimental design, timeline, and representative colonic morphologiesof Apc^(min/+) mice under different treatments. (B) Representativeimages of colon tumor under colonoscopy (upper panels) andrepresentative H&E-stained histological images (lower panels) fromApc^(min/+) mice under different treatments. Scale bar=100 μm. (C)Colonic and small intestine tumor number (upper three panels) and tumorload (lower three panels) of Apc^(min/+) mice under differenttreatments. (D) Immunohistochemistry staining of Ki-67⁺ cells in micecolons with quantitative analysis of Ki-67⁺ index. Results are presentedas mean±S.D. Statistical significance was determined by one-way ANOVA.C.m, C. maltaromaticum; SI, small intestine; W, week.

FIG. 4 shows that C. maltaromaticum protects against intestinaltumorigenesis in azoxymethane (AOM)-induced CRC mice. (A) Schematicdiagram showing the experimental design, timeline, and representativecolonic morphologies of AOM-induced CRC mice model under differenttreatments. (B) Representative images of colon tumor under colonoscopy(upper panels) and representative H&E-stained histological images (lowerpanels) from AOM-induced CRC mice model under different treatments.Scale bar=100 μm. (C) Colonic tumor number (left panel) and tumor load(right panel) of AOM-induced CRC mice under different treatments. (D)Immunohistochemistry staining of Ki-67⁺ cells in mice colons withquantitative analysis of Ki-67⁺ index. Results are presented asmean±S.D. Statistical significance was determined by one-way ANOVA. C.m,C. maltaromaticum; AOM, azoxymethane; W, week.

FIG. 5 shows that C. maltaromaticum preserves gut barrier function,inhibits pro-inflammatory response in Apc^(min/+) mice and AOM-inducedCRC mice. (A) mRNA expression levels of gut barrier-associated genesTjp1 (ZO-1), Ocln, and Cdh1 in colon tissues of Apc^(min/+) mice underdifferent treatments. (B) Expression levels of gut barrier-associatedproteins ZO-1, Occludin, and E-cadherin in colon tissues of Apc^(min/+)mice. (C) LPS concentrations in serum of Apc^(min/+) mice. (D)Representative ultrastructure images of colonic intercellular junctionsof Apc^(min/+) mice. (E) Representative AB-PAS-stained inner mucus layerimages from Apc^(min/+) mice. (F) mRNA expression levels ofpro-inflammatory genes IL-1β, IL-6, Cxcr-2, and anti-inflammatorycytokine IL-10 in colon tissues of Apc^(min/+) mice. (G) mRNA expressionlevels of gut barrier associated genes Tjp1 (ZO-1), Ocln, and Cdh1 incolon tissues of AOM-induced CRC mice under different treatments. (H)Expression levels of gut barrier-associated proteins ZO-1, Occludin, andE-cadherin in colon tissues of AOM-induced CRC mice. (I) RepresentativeAB-PAS-stained inner mucus layer images from AOM-induced CRC. (J) mRNAexpression levels of pro-inflammatory genes IL-1β, IL-6, Cxcr-2, andanti-inflammatory cytokine IL-10 in colon tissues of AOM-induced CRCmice. Results are presented as mean±S.D. Statistical significance wasdetermined by one-way ANOVA. C.m, C. maltaromaticum; AOM, azoxymethane.

FIG. 6 shows that C. maltaromaticum modulates gut microbiota inApc^(min/+) mice. (A) Principal coordinate analysis (PCoA) of speciesrichness of gut microbiota in Apc^(min/+) mice under differenttreatments. (B) Heatmap representation of modulated bacteria at thegenus level in Apc^(min/+) mice. (C) Heatmap representation ofdifferentially abundant bacterial OTUs in Apc^(min/+) mice. Cutoff=|Log2[fold-change (FC)]|>1 & FDR<0.05. Results are presented as mean±S.D.Statistical significance was determined by one-way ANOVA. OUT,Operational taxonomic unit; C.m, C. maltaromaticum.

FIG. 7 shows that C. maltaromaticum increases the abundance of vitamin Dmetabolites in the gut of Apc^(min/+) mice and AOM-induced CRC mice. (A)Principal coordinate analysis (PCoA) for gut metabolomic alteration inApc^(min/+) mice under different treatments. (B) Heatmap representationof differentially metabolites in Apc^(min/+) mice. (C) Pathway analysisof differentially enriched metabolic pathways in Apc^(min/+) mice. (D)Colonic mucosa 1α,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) concentration inApc^(min/+) mice. (E) Levels of enzymes (CYP2R1, CYP27A1, and CYP27b1)responsible for 1,25(OH)₂D₃ biosynthesis in colon tissues of Apc^(min/+)mice. (F) Heatmap representation of enhanced vitamin D metabolites inAOM-induced CRC mice under different treatments. (G) Colonic mucosa1,25(OH)₂D₃ concentration in AOM-induced CRC mice. (H) Levels of enzymes(CYP2R1, CYP27A1, and CYP27b1) responsible 1,25(OH)₂D₃ biosynthesis incolon tissues of AOM-induced CRC mice. Results are presented asmean±S.D. Statistical significance was determined by one-way ANOVA. C.m,C. maltaromaticum; AOM, azoxymethane.

FIG. 8 shows that C. maltaromaticum induces vitamin D signaling inApc^(min/+) mice. (A) Heatmap representation of differentially expressedgenes in colonic tissues of Apc^(min/+) mice under different treatments.(B) mRNA expression levels of bile acid receptors in colon tissues ofApc^(min/+) mice. (C) Gene Set Enrichment Analysis (GSEA) ofdifferentially expressed gene in Apc^(min/+) mice. (D) mRNA (left panel)and protein expression level (right panel) of VDR in colon tissues ofApc^(min/+) mice. (E) mRNA expression level of CRAMP, a VDR target gene,in colon tissues of Apc^(min/+) mice. (F) Immunofluorescence staining ofnuclear VDR⁺ cells in Apc^(min/+) mice colons with quantitative analysisof MFI. (G) mRNA expression level of VDR in TCGA COAD tumor samples. (H)Heatmap representation of expression of VDR target genes in Apc^(min/+)mice. Results are presented as mean±S.D. Statistical significance wasdetermined by one-way ANOVA. MFI, mean fluorescence intensity; C.m, C.maltaromaticum.

FIG. 9 shows that C. maltaromaticum induces vitamin D signaling inAOM-induced CRC mice. (A) mRNA (left panel) and protein expression level(right panel) of VDR in colon tissues of AOM-induced CRC mice. (B) mRNAexpression level of CRAMP in colon tissues of AOM-induced CRC mice. (C)Immunofluorescence staining of nuclear VDR⁺ cells in AOM-induced CRCmice colons with quantitative analysis of MFI. Results are presented asmean±S.D. Statistical significance was determined by one-way ANOVA. MFI,mean fluorescence intensity; C.m, C. maltaromaticum; AOM, azoxymethane.

FIG. 10 shows that C. maltaromaticum inhibits pro-inflammatory responsein Apc^(min/+) mice. (A) Expression of multiple inflammation-relatedgenes in colon tissues of Apc^(min/+) mice under different treatments.(B) Enrichment of multiple inflammation-related pathways in colontissues of Apc^(min/+) mice. Results are presented as mean±S.D.Statistical differentially significance gene expression was determinedas Cutoff=|Log 2(FC)|>1 & FDR<0.05. C.m, C. maltaromaticum.

FIG. 11 shows that L. gallinarum protects against intestinaltumorigenesis in Apc^(Min/+) mice. (A) Schematic diagram showing theexperimental design, timeline of Apc^(Min/+) mouse model. (B)Representative images of colon tumor from Apc^(Min/+) mouse model.Colonoscopy confirmed that colon tumor size in L. gallinarum group wasvisually smaller than tumors in E. coli MG1655 or PBS control groups. Inboth E. coli MG1655 and PBS control groups, colonoscope could not passthrough the tumors. (C) There was no significant difference in bodyweight among three groups of mice during the period of gavage. (D)Number and incidence of bloody stool in L. gallinarum group were lowerthan broth control group after gavage for 8 weeks. (E) Colon tumornumber and tumor size in Apc^(Min/+) mice under different treatments.(F) Small intestinal tumor number and tumor size in Apc^(Min/+) miceunder different treatments. Each black triangle indicates one tumorlocation. P-values are calculated by one-way ANOVA. *p<0.05, **p<0.01,***p<0.001. L.c, L. casei; L.g, L. gallinarum; SI, small intestine.

FIG. 12 shows that L. gallinarum protects against intestinaltumorigenesis in AOM/dextran sulfate sodium (DSS)-induced CRC mice. (A)Schematic diagram showing the experimental design, timeline of maleAOM/DSS mouse model. (B) Representative images of colon tumor from maleAOM/DSS mouse model. (C) Colon tumor number and tumor size in maleAOM/DSS mice under different treatments. Each black triangle indicatesone tumor location. P-values are calculated by one-way ANOVA. *p<0.05,**p<0.01, ***p<0.001. L.c, L. casei; L.g, L. gallinarum; SI, smallintestine.

FIG. 13 shows that L. gallinarum modulates the gut microbiota ofApc^(Min/+) mice. (A) a-diversity analysis of luminal microbiotaoperational taxonomic units (I) at various taxonomic ranks. (B)Principal coordinate analysis (PCoA) of β-diversity based on Bray-Curtisdissimilarity matrix IOTU-level compositional profiles. Ellipsesrepresent 95% confidence intervals. Solid diamond-shaped points in blackdenote species scores, which were calculated using the vegan R-CRANpackage. (C) Heatmap of differentially abundant bacterial OTUs usingone-way analysis of variance, at p<0.05. L.g, L. gallinarum.

FIG. 14 shows that L. gallinarum supernatant inhibits the viability ofcolon cancer cells. The proliferation of cells was measured by MTTassay. Different concentrations of culture supernatant were used forculturing CRC cell lines, HCT116 (5%, 10%, 20%) and LoVo (5%, 10%, 20%),and normal colonic epithelial cell line, NCM460 (5%, 10%, 20%). (A) Theculture supernatant of L. gallinarum, especially in 10% and 20%,significantly suppressed the cell growth of HCT116 from day 4 to day 5.(B) The cell growth of LoVo was also significantly suppressed by theculture supernatant of L. gallinarum with different concentrations; both5% and 10% from day 4 to day 5, and 20% from day 3 to day 5. (C) Nochange in cell growth could be observed in the normal colonic epithelialcell line NCM460. (D) 20% LGCS suppressed colony formation of CRC cells.P-values are calculated by two-way ANOVA. ***p<0.001, ****p<0.0001.ECCS, E. coli culture supernatant; LGCS, L. gallinarum culturesupernatant.

FIG. 15 shows that L. gallinarum supernatant promotes apoptosis insteadof cell cycle arrest in CRC cells. (A) LGCS significantly promotedapoptosis including both early and late phases in two CRC cell linesHCT116, and (B) LoVo, but not (C) in the normal colonic epithelial cellline NCM460. (D) The size and number of CRC patient-derived organoidswas visually reduced in medium containing 10% LGCS. (E) LGCSsignificantly promoted apoptosis including both early and late phases inCRC patient-derived organoids. (F) LGCS had no effect on cell cycledistribution in HCT116, (G) LoVo and (H) NCM460. P-values are calculatedby one-way ANOVA. **p<0.01, ****p<0.0001. ECCS, E. coli culturesupernatant; LGCS, L. gallinarum culture supernatant.

FIG. 16 shows that Anti-tumor molecules produced from L. gallinarum arenon-protein with a molecular weight<3 kDa. (A) Low-molecular-weight(LMW)-LGCS but not HMW-LGCS significantly suppressed cell growth ofHCT116 and LoVo. (B) Decrease in proliferation of CRC cells was observedin heat-inactivated-LGCS. (C) Decrease in proliferation of CRC cells wasobserved in proteinase k-inactivated-LGCS. P-values are calculated bytwo-way ANOVA. *p<0.05, **p<0.01, ****p<0.0001. ECCS, E. coli culturesupernatant; LGCS, L. gallinarum culture supernatant; PK, proteinase K.

FIG. 17 shows that L. gallinarum induces Try metabolism. (A) Score plotsof PCA revealed clear separations of metabolites in culture-supernatantof L. gallinarum, E. coli MG1655 and control broth groups. (B) Heatmapanalysis revealed the abundance of different metabolites in LGCS, ECCSand control broth groups from culture-supernatants. (C) Score plots ofPCA revealed clear separations among L. gallinarum, E. coli MG1655 andcontrol broth treated Apc^(Min/+) mice. (D) Heatmap analysis revealedthe abundance of different metabolites in the gut of Apc^(Min/+) miceunder different treatments. P-values are calculated by Student's t-test.*p<0.05, **p<0.01, ****p<0.0001. ECCS, E. coli culture supernatant;LGCS, L. gallinarum culture supernatant; PK, proteinase K.

FIG. 18 shows that Probio-X catabolizes Try to produce M-X to protectagainst CRC. Targeted metabonomics on L-tryphtophan were performed ondifferent culture supernatants and fecal samples from Apc^(Min/+) miceunder different treatments. (A) Score plots of PCA revealed clearseparations among culture supernatant of Probio-X, E. coli MG1655 andcontrol broth groups. (B) Score plots of PCA revealed clear separationsamong fecal samples from Probio-X-treated, E. coli MG1655-treated andcontrol Apc^(Min/+) mice. (C) Heatmap analysis revealed the abundance ofdifferent metabolites in X.CS, ECCS and control broth groups. (D)Heatmap analysis revealed the abundance of different metabolites in thegut of Apc^(Min/+) mice under different treatments. (E) The cell growthof CRC cells was significantly suppressed by M-X. (F) The cell apoptosisof CRC cells was significantly increased by M-A. (G) Schematic diagramshowing the experimental design, timeline and representative macroscopicimages of colons from of M-A-treated Apc^(Min/+) mouse model. (H) Colon,small intestinal and total tumor number (colon+small intestinal) inApc^(Min/+) mice with or without M-A treatment. (I) Colon, smallintestinal with or without M-A treatment. (J) TUNEL positive stainingcells in colon tissues of Apc^(Min/+) mice with or without M-Atreatment. Each black triangle indicates one tumor location. (K) Thetranscription of AhR was significantly suppressed by CH-223191 (100 nM).(L) Pre-treat CRC cells with CH-223191 (100 nM) for 12 hours abolishedthe anti-proliferation effect of X.CS. (M) Schematic diagram showing theexperimental design, timeline and representative macroscopic images ofcolons from female Apc^(Min/+) mouse model. (N) Colon, small intestinaland total tumor number (colon+small intestinal) in female Apc^(Min/+)mice under different treatments. (0) Colon, small intestinal and totaltumor size (colon+small intestinal) in female Apc^(Min/+) mice underdifferent treatments. (P) Schematic diagram showing the experimentaldesign, timeline and representative macroscopic images of colons frommale Apc^(Min/+) mouse model. (Q) Colon, small intestinal and totaltumor number (colon+small intestinal) in male Apc^(Min/+) mice underdifferent treatments. (R) Colon, small intestinal and total tumor size(colon+small intestinal) in male Apc^(Min/+) mice under differenttreatments. Each black triangle indicates one tumor location. P-valuesare calculated by two-way ANOVA or Student's t-test as appropriate.*p<0.05, **p<0.01, ****p<0.0001. ECCS, E. coli culture supernatant;X.CS, Probio-X culture supernatant; M-X, Metabolite-X.

FIG. 19 shows that Combined L. gallinarum and C. maltaromaticumsynergistically protects against intestinal tumorigenesis inAOM/DSS-induced CRC mice. (A) Schematic diagram showing the experimentaldesign, timeline of AOM/DSS mouse model. (B) Representative images ofcolon tumor from AOM/DSS mouse model. (C) Colon tumor number and tumorsize in male AOM/DSS mice under different treatments. Each blacktriangle indicates one tumor location. P-values are calculated byone-way ANOVA. *p<0.05, **p<0.01, ***p<0.001.C.m, C. maltaromaticum;L.g, L. gallinarum.

FIG. 20 shows that Combined L. gallinarum and C. maltaromaticumsynergistically modified the tumor immune microenvironment inAOM/DSS-induced CRC mice.

FIG. 21 shows that L. lactis protects against intestinal tumorigenesisin Apc^(Min/+) mice. (A) Schematic diagram showing the experimentaldesign, timeline, and representative colonic morphologies of Apc^(min/+)mice under different treatments. (B) Representative images of colontumor under colonoscopy (upper panels) and representative H&E-stainedhistological images (lower panels) from Apc^(min/+) mice under differenttreatments. (C) Total tumor number and tumor load of Apc^(min/+) miceunder different treatments. Results are presented as mean±S.D.Statistical significance was determined by one-way ANOVA. L.L, L.lactis; W, week.

FIG. 22 shows that L. lactis conditioned medium (LL.CM) inhibits theviability of colon cancer cells. The proliferation of cells was measuredby MTT assay. (A) LL.CM at 5% concentration significantly suppressed theCRC cells (HCT116 and HT29) growth but not the normal colonic epithelialcell line NCM460. (B) 5% LL.CM suppressed patient-derived organoids'size significantly. P-values are calculated by two-way ANOVA or one-wayANOVA where appropriate. * ****p<0.0001. Ec.CM, E. coli conditionedmedium; LL.CM, L. lactis conditioned medium.

FIG. 23 shows that anti-tumor molecules produced from L. lactis areprotein(s)>100 kDa in molecular weight. (A) Decrease in proliferation ofCRC cells was not observed in heat- and PK-inactivated-LL.CM. (B)Decrease in colony formation of CRC cells was not observed in heat- andPK-inactivated-LL.CM. (C) Decrease in proliferation of CRC cells wasonly observed in LL.CM>100 kDa. P-values are calculated by two-way ANOVAor one-way ANOVA where appropriate. ****p<0.0001. Ec.CM, E. coliconditioned medium; LL.CM, L. lactis conditioned medium.; PK, proteinaseK.

FIG. 24 shows that anti-tumor molecules produced from L. lactis containaminopeptidase. L.L, L. lactis.

DEFINITIONS

In this disclosure the terms “colorectal cancer (CRC)” and “coloncancer” have the same meaning and refer to a cancer of the largeintestine (colon), the lower part of human digestive system, althoughrectal cancer often more specifically refers to a cancer of the lastseveral inches of the colon, the rectum. A “colorectal cancer cell” is acolon epithelial cell possessing characteristics of colon cancer andencompasses a precancerous cell, which is in the early stages ofconversion to a cancer cell or which is predisposed for conversion to acancer cell. Such cells may exhibit one or more phenotypic traitscharacteristic of the cancerous cells.

The term “indole-3-lactic acid” or ILA is used herein to refer to asmall molecule that is a metabolite of tryptophan, secreted by certainbacterial species, having the chemical structure shown below:

As used here, “culture supernatant” refers to the aqueous portion of abacterial culture that has been placed under suitable conditions (e.g.,at about 28 to 37° C. for 12-24 hours) permitting log phaseproliferation of the bacterial cells and subsequently has the bacterialcells substantially removed, e.g., by centrifugation for at least about5 minutes, such as about 5-10 minutes, at about 5,000 or higher rpm,such as about 5,000-10,000 rpm. Also encompassed in the concept of abacteria culture supernatant are aqueous products that have been subjectto dilution or concentration or fractionation based on the molecularweight of compounds present in the supernatant.

As used in this application, an “increase” or a “decrease” refers to adetectable positive or negative change in quantity from a comparisoncontrol, e.g., an established standard control. An increase is apositive change that is typically at least 10%, or at least 20%, or 50%,or 100%, and can be as high as at least 2-fold or at least 5-fold oreven 10-fold of the control value. Similarly, a decrease is a negativechange that is typically at least 10%, or at least 20%, 30%, or 50%, oreven as high as at least 80% or 90% of the control value. Other termsindicating quantitative changes or differences from a comparative basis,such as “more,” “less,” “higher,” and “lower,” are used in thisapplication in the same fashion as described above. In contrast, theterm “substantially the same” or “substantially lack of change”indicates little to no change in quantity from the standard controlvalue, typically within ±10% of the standard control, or within ±5%, 2%,1%, or even less variation from the standard control.

The term “inhibiting” or “inhibition,” as used herein, refers to anydetectable negative effect on a target biological process, such asRNA/protein expression of a target gene, the biological activity of atarget protein, cellular signal transduction, cell proliferation,presence/level of an organism especially a micro-organism, anymeasurable biomarker, bio-parameter, or symptom in a subject, and thelike. Typically, an inhibition is reflected in a decrease of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater in the targetprocess (e.g., a subject's bodyweight, or the blood glucose/cholesterollevel, or any measurable symptom or biomarker in a subject, such as aninfection rate among subjects by a pathogenic infectious agent or adisease incidence), or any one of the downstream parameters mentionedabove, when compared to a control. “Inhibition” further includes a 100%reduction, i.e., a complete elimination, prevention, or abolition of atarget biological process or signal or disease/symptom. The otherrelative terms such as “suppressing,” “suppression,” “reducing,” and“reduction” are used in a similar fashion in this disclosure to refer todecreases to different levels (e.g., at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater decrease compared to a control level) upto complete elimination of a target biological process or signal ordisease/symptom. On the other hand, terms such as “activate,”“activating,” “activation,” “increase,” “increasing,” “promote,”“promoting,” “enhance,” “enhancing,” or “enhancement” are used in thisdisclosure to encompass positive changes at different levels (e.g., atleast about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,or greater such as 3, 5, 8, 10, 20-fold increase compared to a controllevel in a target process, signal, or symptom/disease incidence.

As used herein, the term “treatment” or “treating” includes boththerapeutic and preventative measures taken to address the presence of adisease or condition or the risk of developing such disease or conditionat a later time. It encompasses therapeutic or preventive measures foralleviating ongoing symptoms, inhibiting or slowing disease progression,delaying of onset of symptoms, or eliminating or reducing side-effectscaused by such disease or condition. A preventive measure in thiscontext and its variations do not require 100% elimination of theoccurrence of an event; rather, they refer to a suppression or reductionin the likelihood or severity of such occurrence or a delay in suchoccurrence.

The term “severity” of a disease refers to the level and extent to whicha disease progresses to cause detrimental effects on the well-being andhealth of a patient suffering from the disease, such as short-term andlong-term physical, mental, and psychological disability, up to andincluding death of the patient. Severity of a disease can be reflectedin the nature and quantity of the necessary therapeutic and maintenancemeasures, the time duration required for patient recovery, the extent ofpossible recovery, the percentage of patient full recovery, thepercentage of patients in need of long-term care, and mortality rate.

A “patient” or “subject” receiving the composition or treatment methodof this invention is a human, including both adult and juvenile human,of any age, gender, and ethnic background, who may not have beendiagnosed with any particular disease or disorder (e.g., colon cancer orCRC) but is in need of prophylactic or therapeutic treatment for thedisease. Typically, the patient or subject receiving treatment accordingto the method of this invention to prevent or ameliorate CRC or any ofits symptoms is not otherwise in need of treatment by the sametherapeutic agents. For example, if a subject is receiving the probioticcomposition according to the claimed method, the subject is notsuffering from any disease that is known to be treated by the sametherapeutic agents. Although a patient may be of any age, in some casesthe patient is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 yearsof age; in some cases, a patient may be between 40 and 45 years old, orbetween 50 and 65 years of age, or between 65 and 85 years of age. A“child” subject is one under the age of 18 years, e.g., about 5 to 17, 9or 10 to 17, or 12 to 17 years old, including an “infant,” who isyounger than about 12 months old, e.g., younger than about 10, 8, 6, 4,or 2 months old, whereas an “adult” subject is one who is 18 years orolder.

The term “effective amount,” as used herein, refers to an amount thatproduces the intended (e.g., therapeutic or prophylactic) effects forwhich a substance is administered. The effects include the prevention,correction, or inhibition of progression of the symptoms of a particulardisease/condition and related complications to any detectable extent,e.g., incidence of disease and related disorder (e.g., CRC). The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see,e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd,The Art, Science and Technology of Pharmaceutical Compounding (1999);and Pickar, Dosage Calculations (1999)).

The term “about” when used in reference to a given value denotes a rangeencompassing ±10% of the value. For instance, “about 10” encompasses therange of 9 to 11.

A “pharmaceutically acceptable” or “pharmacologically acceptable”excipient is a substance that is not biologically harmful or otherwiseundesirable, i.e., the excipient may be administered to an individualalong with a bioactive agent without causing any undesirable biologicaleffects. Neither would the excipient interact in a deleterious mannerwith any of the components of the composition in which it is contained.

The term “excipient” refers to any essentially accessory substance thatmay be present in the finished dosage form of the composition of thisinvention. For example, the term “excipient” includes vehicles, binders,disintegrants, fillers (diluents), lubricants, glidants (flowenhancers), compression aids, colors, sweeteners, preservatives,suspending/dispersing agents, film formers/coatings, flavors andprinting inks.

The term “consisting essentially of,” when used in the context ofdescribing a composition containing an active ingredient or multipleactive ingredients, refers to the fact that the composition does notcontain other ingredients possessing any similar or relevant biologicalactivity of the active ingredient(s) or capable of enhancing orsuppressing the activity, whereas one or more inactive ingredients suchas physiological or pharmaceutically acceptable excipients may bepresent in the composition. For example, a composition consistingessentially of active agents (for instance, a combination of L.gallinarum and C. maltaromaticum) effective for treating or preventingCRC in a subject is a composition that does not contain any other agentsthat may have any detectable positive or negative effect on the sametarget process (e.g., inhibition of the onset or progression oftumorigenesis of the colon) or that may increase or decrease to anymeasurable extent of the disease severity among the receiving subjects.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Using fecal shotgun metagenomic sequencing, the present inventors havepreviously identified Lactobacillus gallinarum as one of the mostdepleted probiotic species in the stool of colorectal cancer (CRC)patients. This study was intended to determine the potentialanti-tumorigenic role of L. gallinarum, optionally in combination withanother one or two bacterial species (such as Lactococcus lactis orCarnobacterium maltaromaticum), in colorectal tumorigenesis.

Specifically, Apc^(min/+) mice and azoxymethane/dextran sulfatesodium-treated mice were gavaged with L. gallinarum, E. coli MG1655(control bacteria), or plain culture broth daily until the developmentof neoplastic lesions. Intestinal tumor numbers and sizes weredetermined after sacrifice. CRC organoids and CRC cell lines (HCT116 andLoVo) were cultured with L. gallinarum or E. coli MG1655culture-supernatant to evaluate cell proliferation, apoptosis and cellcycle distribution. Gut microbiota was assessed by 16S rRNA sequencing.Anti-tumor molecule produced from L. gallinarum was identified by liquidchromatography mass spectrometry (LC-MS/MS) and targeted massspectrometry assay.

L. gallinarum significantly reduced intestinal tumor number and sizecompared with E. coli MG1655 and plain culture broth in both male andfemale murine intestinal tumorigenesis models. Fecal microbial profilingrevealed the enrichment of probiotics and depletion of pathogenicbacteria in L. gallinarum-treated mice. Culturing CRC cells with L.gallinarum culture-supernatant (5%, 10% and 20%)concentration-dependently suppressed cell proliferation and colonyformation. L. gallinarum culture-supernatant significantly promotedapoptosis in CRC cells and patient-derived organoids, but not in normalcolon epithelial cells. Only L. gallinarum culture-supernatant withfraction size<3 kDa suppressed cell proliferation in CRC cells. UsingLC-MS/MS, L-Tryptophan was identified to be increased in both of the L.gallinarum culture-supernatant and L. gallinarum-treated mice gut.Further high throughput tryptophan targeted metabonomics revealed thatindole-3-lactic acid (ILA) was one of the most significantly increasedlow-molecular-weight metabolite (<3 kDa) secreted by L. gallinarum.

In summary, this study provides the following observations: (1) L.gallinarum inhibited colorectal tumorigenesis in Apc^(min/+) mice and inazoxymethane/dextran sulfate sodium-treated mice; (2) L. gallinarumincreased the abundance of gut probiotics and depleted potential gutpathogens; (3) L. gallinarum culture-supernatant suppressed cellproliferation and induced apoptosis in patient-derived organoids and inCRC cell lines; and (4) Non-protein secreted molecule(s) with amolecular weight<3 kDa from L. gallinarum mediated the anti-CRC effect.Indole-3-lactic acid, a small molecule with known anti-inflammatoryproperty, was identified as the most enriched metabolite secreted by L.gallinarum. Similarly, Lactococcus lactis and at least one highmolecular weight (>100 kDa) protein with aminopeptidase activity in theL. lactis culture have been shown to possess a suppressive activity onthe CRC cells. It is therefore concluded that L. gallinarum as well asL. lactis protects against intestinal tumorigenesis by producingprotecting metabolites that can promote apoptosis on CRC cells.

II. Pharmaceutical Compositions and Administration

The present invention provides pharmaceutical compositions comprising aneffective amount of live bacteria L. gallinarum or L. lactis, ILA, or anaminopeptidase produced by L. lactis with a >100 kDa molecular weight,optionally in further combination with live bacteria C. maltaromaticum,for administration to a person to reduce the risk of later developingCRC or to treat CRC a person already suffers from. Pharmaceuticalcompositions of the invention are suitable for use in a variety of drugdelivery systems. Suitable formulations for use in the present inventionare found in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Philadelphia, Pa., 17th ed. (1985). For a brief review ofmethods for drug delivery, see, Langer, Science 249: 1527-1533 (1990).

The pharmaceutical compositions of the present invention can beadministered by various routes, e.g., systemic administration via oralingestion or local delivery using a rectal suppository. The preferredroute of administering the pharmaceutical compositions is oraladministration at daily doses of about 10⁸ to about 10¹² CFU for livebacteria L. gallinarum or L. lactis, or a combination of live bacteriaL. gallinarum (or L. lactis) and C. maltaromaticum, at a ratio among anytwo of the bacteria species ranging from about 1:5, 1:4, 1:3, 1:2, 1:1,2:1, 3:1, 4:1, to about 5:1. These two or three species of bacteria maybe administered either in one single composition or in multiplecompositions. Optionally, the small molecule of ILA, a downstreamcatabolite of L-tryptophan (Try) secreted by L. gallinarum, or anaminopeptidase produced by L. lactis with a molecular weight of greaterthan 100 kDa, is administered to the subject in lieu of live bacteria L.gallinarum or L. lactis. Typically, ILA or the aminopeptidase may bepresent in the composition for administration in the range of about 0.5to about 50 or 100, or about 1 to about 20 or 25, or about 2 to about 10or 15, or about 5 to about 10 mg per kg patient bodyweight. As a furtheralternative, L. gallinarum or L. lactis culture supernatant, which mayhave been further processed, e.g., fractionated to capture molecularweight range of less than about 3 kDa or greater than about 100 kDa,respectively.

For preparing pharmaceutical compositions containing live bacteria L.gallinarum or L. lactis, ILA, or the L. lactis—produced aminopeptidasewith a molecular weight greater than about 100 kDa, optionally incombination with live bacteria C. maltaromaticum, one or more inert andpharmaceutically acceptable carriers are used. The pharmaceuticalcarrier can be either solid or liquid. Solid form preparations include,for example, powders, tablets, dispersible granules, capsules, cachets,and suppositories. A solid carrier can be one or more substances thatcan also act as diluents, flavoring agents, solubilizers, lubricants,suspending agents, binders, or tablet disintegrating agents; it can alsobe an encapsulating material.

In powders, the carrier is generally a finely divided solid that is in amixture with the finely divided active component, e.g., L. gallinarum,L. lactis, ILA, or the L. lactis-secreted aminopeptidase with amolecular weight of greater than about 100 kDa, optionally further incombination with C. maltaromaticum. In tablets, the active ingredient ismixed with the carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.

For preparing pharmaceutical compositions in the form of suppositories,a low-melting wax such as a mixture of fatty acid glycerides and cocoabutter is first melted and the active ingredient is dispersed thereinby, for example, stirring. The molten homogeneous mixture is then pouredinto convenient-sized molds and allowed to cool and solidify.

Powders and tablets preferably contain between about 5% to about 100% byweight of the active ingredient(s) (e.g., L. gallinarum, L. lactis, ILA,or the L. lactis-secreted aminopeptidase with a molecular weight of >100kDa, optionally further in combination with C. maltaromaticum). Suitablecarriers include, for example, magnesium carbonate, magnesium stearate,talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methylcellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoabutter, and the like.

The pharmaceutical compositions can include the formulation of theactive ingredient(s) e.g., L. gallinarum, L. lactis, ILA, or the L.lactis-secreted aminopeptidase with a molecular weight of >100 kDa,optionally further in combination with C. maltaromaticum, withencapsulating material as a carrier providing a capsule in which theactive ingredient(s) (with or without other carriers) is surrounded bythe carrier, such that the carrier is thus in association with theactive ingredient(s). In a similar manner, sachets can also be included.Tablets, powders, sachets, and capsules can be used as solid dosageforms suitable for oral administration.

Liquid pharmaceutical compositions include, for example, solutionssuitable for oral administration or local delivery, suspensions, andemulsions suitable for oral administration. Sterile water solutions ofthe active component (e.g., L. gallinarum, L. lactis, ILA, or the L.lactis-secreted aminopeptidase with a molecular weight of >100 kDa,optionally further in combination with C. maltaromaticum) or sterilesolutions of the active component in solvents comprising water, bufferedwater, saline, PBS, ethanol, or propylene glycol are examples of liquidor semi-liquid compositions suitable for oral administration or localdelivery such as by rectal suppository. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents, and thelike.

Sterile solutions can be prepared by dissolving the active component(e.g., L. gallinarum, L. lactis, ILA, or the L. lactis-secretedaminopeptidase with a molecular weight of >100 kDa, optionally furtherin combination with C. maltaromaticum) in the desired solvent system,and then passing the resulting solution through a membrane filter tosterilize it or, alternatively, by dissolving the sterile activecomponent in a previously sterilized solvent under sterile conditions.The resulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the preparationstypically will be between 3 and 11, more preferably from 5 to 9, andmost preferably from 7 to 8.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of an active agent sufficient to effectively enhance theefficacy of a vaccine and/or reduce or eliminate undesirable adverseeffects of a vaccine.

III. Kits

The invention also provides kits for reducing the risk of laterdeveloping CRC or for inhibiting progression of the disease/reducingseverity of the disease symptoms in an individual according to themethod disclosed herein. The kits typically include a plurality ofcontainers, each containing a composition comprising one or more of theactive agents, such as live bacteria L. gallinarum, L. lactis, and C.maltaromaticum, or one or more proteins secreted by the bacteria, e.g.,ILA or an aminopeptidase produced by L. lactis having a >100 kDamolecular weight. For example, a first container contains an effectiveamount of live bacteria L. gallinarum or L. lactis, and a secondcontainer contains an effective amount of live bacteria C.maltaromaticum. As another example, a first container contains aneffective amount of ILA or the aminopeptidase, or supernatant of an L.gallinarum or L. lactis culture, and a second container contains aneffective amount of live bacteria C. maltaromaticum. As an additionalexample, the kits include one single container contains live bacteria L.gallinarum (or L. lactis) and live bacteria C. maltaromaticum,combinedly in an effective amount for the intended purpose. Further,additional agents or drugs that are known to be therapeuticallyeffective for prevention and/or treatment of the disease, including forameliorating the symptoms and reducing the severity of the disease, aswell as for facilitating recovery from the disease may be included inthe kit. The plurality of containers of the kit each may contain adifferent active agent/drug or a distinct combination of two or more ofthe active agents or drugs. The kit may further include informationalmaterial providing instructions on how to dispense the pharmaceuticalcomposition(s), including description of the type of patients who may betreated, e.g., human patients who have been deemed as with high risk ofdeveloping the disease, for example, due to genetic predisposition,family history of cancer (especially colon cancer), and/or personaltraits and medical background such as age (50 years or older), gender(male), diabetes mellitus, obesity, and inflammatory bowel disease, aswell as smoking and certain dietary choices, e.g., inadequate intake offiber, high consumption of alcohol, red meat, and high salt or high fator preserved foods, as well as the type of patients not to be includedin the claimed method, e.g., those who have been diagnosed with apre-existing condition, such as colitis, that already requires theadministration of the active components such as L. gallinarum, L.lactis, ILA, the L. lactis-secreted aminopeptidase with a molecularweight of >100 kDa, or C. maltaromaticum), the dosage, frequency, andmanner of administration, and the like.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example I: Carnobacterium maltaromaticum Modulates the Gut Microbiotaand Enhances Vitamin D-Related Metabolites to Protect Against ColorectalTumorigenesis Background of the Invention

Colorectal cancer (CRC) is one of the most commonly diagnosed and deadlymalignancies worldwide. Along with genetic, epigenetic, andenvironmental factors, the role of gut microbiota in the initiation andprogression of CRC has been recognized in the past decades¹. Thedysbiosis of microbes during the long, stepwise progression ofintestinal tumorigenesis enabled possible CRC prevention throughmaintaining intestinal microbial balance by consumption of CRC-depletedprobiotics and its metabolites².

So far, probiotics-based therapeutics have aroused interest from themedical community given their high safety profile. Administration ofprobiotics, such as Lactobacillus casei (strain BL23), Clostridiumbutyricum, and Bifidobacterium bigidum, have been found to re-establishthe gut microbial balance by diminishing the colonization of pathogens³.Clinical studies also showed oral administration of Bifidobacterium andLactobacillus probiotics could mitigate dysbiosis in CRC patients,thereby supporting the possibility of using probiotics as prophylacticsfor maintaining healthy microbiota states with minimal side effects⁴. Inaddition to the microbial interactions, health-promoting metabolitessecreted from probiotics could exert direct anti-cancer effects⁵. Themetagenomic profiling from previous studies has demonstrated thatStreptococcus thermophilus, a CRC-depleted probiotic, could preventintestinal tumorigenesis through secreting β-galactosidase⁶. Thesefindings support the importance of determining the tumor-suppressingeffect of other CRC-depleted bacteria to provide new strategies for CRCprevention.

Using shotgun metagenomic sequencing of 526 multi-cohort fecal samplesfrom 255 CRC patients and 271 healthy controls, the present inventorshave identified a probiotic species, namely Carnobacteriummaltaromaticum (C. maltaromaticum), being significantly depleted in thestool of CRC patients. Here, the functional role of C. maltaromaticum inmitigating colorectal tumorigenesis in mouse models and cultured CRCcells is elucidated. This tumor-suppressing effect has been revealed tobe attributed to the activated biosynthetic pathway of vitamin D.

Materials and Methods

C. maltaromaticum Quantification in Human Fecal Samples

Human stool samples were collected form 78 adults and the fecal DNAswere extracted as described previously. Amplification and detection ofC. maltaromaticum were conducted using Universal SYBR Green Masterreaction, and the reaction was analyzed by QuantStudio™ 7 Flex System(Thermo Fisher Scientific Waltham, Mass.). This study protocol wasapproved by the Joint CUHK-NTEC Clinical Research Ethics Committee.Written informed consents were obtained from individuals.

Bacteria Strains and Growth Conditions

C. maltaromaticum (ATCC B270) and Escherichia coli strain MG1655 (ATCC700926) were purchased from American Type Culture Collection (ATCC;Manassas, Va.). They were cultured in Brain Heart Infusion (BHI) broth(CM1135B; Thermo Fisher Scientific, West Palm Beach, Fla.) at 37° C.under aerobic condition for 24 hours before use.

Cell Culture

Colon cancer cell lines HCT116 and DLD1 were obtained from American TypeCulture Collection (ATCC). Normal colonic epithelial cell line NCM460was obtained from INCELL Corporation (San Antonio, Tex.). All the celllines were cultured in high-glucose Dulbecco's Modified Eagle's Medium(DMEM) (Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetalbovine serum (FBS) (Thermo Fisher Scientific), 1%penicillin/streptomycin in a humidified atmosphere containing 5% CO₂.For co-culture of epithelial cells with bacteria, thepenicillin/streptomycin in the culture medium was removed and cells wereexposed to bacteria with a multiplicity of infection (MOI) of 200 for 2hours. Cell viability and cell cycle were determined in the same way aswe reported previously.

Adenomatous Polyposis Coli/Multiple Intestinal Neoplasia CRC Model

C57BL/6J-ApcMin/J mice, which develop intestinal polyps spontaneouslywas used as a model of spontaneous intestinal neoplasia. They werepurchased from the Jackson Laboratory (Bar Harbor, Me., USA) andmaintained in the animal facility at the Chinese University of HongKong. Mice at 4-5 weeks old were divided into 3 groups—1×10⁸ colonyforming units (CFU) of C. maltaromaticum or E. coli MG1655, or the samevolume of BHI was gavaged to them once daily for 12 weeks. Colorectaltumor formation was monitored by mouse colonoscopy (Coloview, KarlStroz, Germany). Stool samples were collected weekly.

Carcinogen-Induced Colon Cancer Model

5-week-old male conventional C57BL/6 wild-type mice were subject to 6consecutive injections of azoxymethane (AOM; 10 mg/kg, intraperitonealinjection) at 1-week intervals to induce sporadic CRC. The sametreatment regimen with the Apc^(min/+) mice was used in this AOM model.Mice were raised to 26 weeks for the evaluation of the probiotictreatment efficacy. All experimental procedures adhered to theguidelines approved by the Animal Ethics Committee of the ChineseUniversity of Hong Kong.

16S rRNA Gene Sequencing

Mice fecal DNA extraction and purification were performed by usingQuick-DNA™ Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine,Calif.). DNA library was constructed on Illumina MiSeq platform asdescribed in our previous study⁷. University primers 341F (SEQ IDNO:1:5′-CCTAYGGGRBGCASCAG-3′) and 806R (SEQ ID NO:2:5′-GGACTACNNGGGTATCTAAT-3′) targeting 16S rRNA genes V3-V4 hypervariableregions were used for sequencing. Bacteria OUTs with P<0.05 and |log2[fold change (FC)]|>1 (FC>2 or FC<0.5) were deemed statisticallysignificant.

Metabolomics Profiling and Metabolites Analysis

Metabonomics was performed by BIOTREE, Shanghai, China. The metaboliteswere transferred to a fresh glass vial for liquid chromatography-tandemmass spectrometry (LC-MS/MS) analysis. Ultra-high performance liquidchromatography (UHPLC) separation was carried out using a 1290 Infinityseries UHPLC System (Agilent Technologies, Palo Alto, Calif.), equippedwith a UPLC BEH Amide column. The TripleTOF 6600 mass spectrometry (ABSciex, Foster City, Calif.) was used to acquire tandem mass spectrometry(MS/MS) spectra on an information-dependent basis during LC-MS/MSexperiment. The metabolite identification was based on in-house MS2database, Human Metabolome Database (HMDB, see website: hmdb.ca), andMETLIN metabolite database (see website: metlin.scripps.edu).

Vitamin D Concentration in Colonic Mucosa

Colonic samples were washed in PBS and dissected in extraction buffer(2:1 methanol:methylene chloride) prior to homogenization. Samples werethen vortexed and centrifuged at 10,000×g for 10 minutes. Thesupernatant was carefully collected and evaporated under a gentle streamof nitrogen at 37° C. The dried samples were reconstituted with ELISAbuffer and the 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) level wasmeasured by using Vitamin D ELISA Kit (Cayman, Mich., USA) following themanufacture's instruction. The remaining protein was re-extracted withradioimmunoprecipitation assay buffer (RIPA) for vitamin D concentrationnormalization.

Reverse Transcription-Quantitative PCR (RT-qPCR)

Total RNAs were extracted from cell pellets or colonic tissues usingTRIzol Reagent (Life Technologies). Complementary DNA (cDNA) wasprepared using PrimeScript RT Reagent Kit with gDNA Eraser (Takara,Shiga, Japan). The relative level of specific genes was determined byQuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher Scientific).

Western Blot

Total protein from cell pallets or colonic tissues was isolated andseparated by SDS-PAGE (6%-12%). The protein in SDS-PAGE was thentransferred onto polyvinylidene difluoride (PVDF) membranes (EMDMillipore, Billerica, Mass., USA) for about 1-2 hours, which was thenblocked with 10% non-fat milk in 0.05% Tris-based saline-Tween 20 for 2hours at room temperature. The membrane was incubated with primaryantibodies overnight at 4° C. and then with secondary antibody at roomtemperature for 1 hour. The protein band intensities were detected byECL Plus Western Blotting Detection Reagents (GE Healthcare).

Transmission Electron Microscopy (TEM)

Colonic tissues were dissected into small pieces and fixed in 2.0%glutaraldehyde in 0.1M sodium cacodylate (Electron Microscopy Sciences,Hatfield, Pa.). Ultrathin sections were prepared on a Reichert UltracutE ultramicrotome. The ultrastructure images of the tissues were acquiredusing a Philips CM100 TEM.

Colonic Permeability Assay

Colonic permeability was determined by measuring the levels oflipopolysaccharides (LPS) in serum by a mouse LPS enzyme-linked immunesorbent assay (ELISA) Kit (Cusabio, Wuhan, China) according to themanufacturer's instruction.

Alcian Blue-Periodic Acid Schiff (AB-PAS) Staining

Carnoy's solution-fixed colonic tissues were prepared for AB-PASstaining as we described previously⁸. Briefly, the mucus-containingcolon sections were stained purple-red. The thickness of themucus-secreting layer was measured perpendicularly to the mucosalsurface from the edge of the epithelium to the outermost part of themucus-secreting layer under microscopy at 100× magnification. Fiverandom microscopic fields were counted for each sample.

Ki-67 Immunohistochemistry (IHC) Staining

Paraffin-embedded colon sections were used for Ki-67 (Abcam, 16667) IHCstaining. The proportion of Ki-67 positive cells was used fordetermining the cell proliferation index. One thousand cells in fiverandom microscopic fields were counted for each sample.

VDR Immunofluorescence (IF) Staining

Paraffin-embedded colon sections were deparaffinized, antigen-retrieved,blocked, and incubated with primary VDR antibody (Cell signaling,12550). The mean fluorescence intensity (MFI) of nuclear VDR wasmeasured under a laser scanning confocal microscope (LEICA TCS SP8,Wetzlar, Germany).

Gene Expression Profiling from RNA-Seq in the Cancer Genome Atlas (TCGA)Dataset

The gene expression data of colon adenocarcinoma samples were retrievedfrom The Cancer Genome Altas (TCGA) database using the TCGAbiolinks Rpackage⁹. The unaligned RNA-seq data in fastq format of colonadenocarcinoma were downloaded from The Cancer Genome Altas (TCGA)database¹⁰. The pathseq¹¹ pipeline was used to map those unalignedRNA-seq reads to gut-related bacteria after subtraction of human readsand low quality. The pre-built host genome was obtained from the GATKResource Bundle FTP server in /bundle/pathseq/. The microbe referencesused here included 1520 cultivated bacterial genomes¹² and also coloncancer related bacteria which were identified through extensive andstatistically-rigorous validation^(7,13). The normalized score generatedby pathseq was used as a relative transcriptome abundance for eachspecies. This study meets the publication guidelines provided by TCGA(see detailed description at website:cancergenome.nih.gov/publications/publicationguidelines).

Statistical Analysis

Values are expressed as mean±standard deviation (SD) for both in vivoand in vitro experiments. Comparisons between two groups were performedusing a two-sided Student's t-test. Analysis of variance (ANOVA) wasused to compare differences among multiple groups, and post-hoc analysiswas performed by Tukey's multiple comparisons test. P-values<0.05indicate statistical significance.

Results

C. maltaromaticum is Depleted in Stool Samples of Patients with CRC

To further verify the reduced abundance of C. maltaromaticum in CRC,qPCR was performed with stool samples from 39 patients with CRC and 39healthy individuals. As shown in FIG. 1A, C. maltaromaticum wassignificantly depleted in stool samples from patients with CRC ascompared with those from normal subjects (P=0.0055). Survivalprobability data from TCGA further revealed that the high abundance ofC. maltaromaticum was associated with a better survival of CRC patients(FIG. 1B), indicating the possible anti-CRC effect of C. maltaromaticum.

C. maltaromaticum Inhibits the Viability of Colon Cancer Cells

To investigate the direct tumor-suppressive effect of C. maltaromaticumin vitro, CRC cell lines DLD1 and HCTLL6, and colon normal epithelialcell line NCM460, were co-incubated with C. maltaromaticum at MOI of100, 200 and 400 for 2 hours in aerobic condition, respectively. E. colistrain MG1655 was used as bacteria control. It was discovered that C.maltaromaticum significantly decreased the number of viable CRC cells,but not the normal colonic epithelial cells at MOI of 200 (FIG. 2A-2C).In keeping with this, C. maltaromaticum treatment retarded cell cycleprogression of CRC cells in the G2/M phase at MOI of 200 (FIG. 2D).These results indicate that the CRC-depleted C. maltaromaticum couldsuppress the CRC cell proliferation directly.

C. maltaromaticum Protects Against Intestinal Tumorigenesis inApc^(min/+) Mice

To validate the suppressive effect of C. maltaromaticum on colorectaltumorigenesis in vivo, 200 μl of bacterial suspension containing 1×10⁸CFU of C. maltaromaticum was orally gavaged to 5-week-old Apc^(min/+)mice daily for 12 consecutive weeks. The same volume of BHI or sameamount of E. coli (strain MG1655) were used as control and administratedto the mice the same way as C. maltaromaticum (FIG. 3A). Mousecolonoscopy was performed before harvest. Visually reduced tumor sizewas observed in C. maltaromaticum-gavaged mice as compared with E. colistrain MG1655 or BHI treatment (FIG. 3B, upper). After sacrifice,significantly reduced tumor number and tumor load were observed in bothcolon and small intestine of C. maltaromaticum treated Apc^(min/+) mice(FIG. 3C). The tumor histology was further examined (FIG. 3B, lower),and the decreased proportion of Ki-67⁺ colonic epithelial cells was alsoobserved in C. maltaromaticum-treated Apc^(min/+) mice (FIG. 3D) ascompared with both BHI- and E. coli strain MG1655-treated group,indicating that C. maltaromaticum could protect against intestinaltumorigenesis in Apc^(min/+) mice.

C. maltaromaticum Protects Against Intestinal Tumorigenesis inAOM-Induced CRC Mice

To further verify the CRC-suppressive effect of C. maltaromaticum, anAOM-induced CRC model was established, in which 5-week-old C57BL/6 micewere injected with the carcinogen AOM (10 mg/kg) once a week for 6weeks, followed by oral administration of C. maltaromaticum, E. colistrain MG1655 or BHI for 26 weeks (FIG. 4A). Visual reduction of tumorsize was observed in C. maltaromaticum-treated mice during colonoscopy(FIG. 4B, upper). The tumor number and tumor load were also decreased inC. maltaromaticum-treated mice (FIG. 4C). Histological occurrence of CRCwas further confirmed (FIG. 4B, lower) and the Ki-67 positive cells werealso found to be decreased in C. maltaromaticum-treated mice (FIG. 4D).These findings indicated that C. maltaromaticum could also suppressintestinal tumorigenesis in AOM-induced CRC mice.

C. maltaromaticum Preserves Gut Barrier Function and InhibitsPro-Inflammatory Response in Murine Models of CRC

Gut barrier dysfunction and its related inflammatory response are knownto be associated with the initiation and progression of CRC³. To assessthe involvement of C. maltaromaticum in modulating gut barrier function,the mRNA and protein expression levels of key tight junction genes andproteins were examined by RT-qPCR and Western blots, respectively.Administration of C. maltaromaticum markedly increased the colonic tightjunction regulators, including Tjp1 (ZO-1), Ocln (Occludin), and Cdh1(E-cadherin) at mRNA (FIG. 5A) and protein (FIG. 5B) level inApc^(min/+) mice. The leakage of gut bacterial lipopolysaccharides (LPS)into the systemic circulation was then measured for inferring the gutbarrier function. It was discovered that Apc^(min/+) mice gavaged withC. maltaromaticum had a significantly lower serum level of LPS (FIG.5C). Visualization of cell-cell junctions with TEM further indicatedthat C. maltaromaticum could shorten the junction width (FIG. 5D). Inkeeping with this, the thickness of the inner mucus layer stained byAB-PAS confirmed the increased thickness of the mucus-containing layerin C. maltaromaticum-treated Apc^(min/+) mice (FIG. 5E). To examine therole of C. maltaromaticum in modulating the inflammatory response, thesoluble factors' expression level in colonic tissues was measured. Ascompared with BHI and E. coli strain MG1655 treatment mice,administration of C. maltaromaticum significantly attenuated theexpression of key pro-inflammatory genes, including interleukin (IL)-1β,IL-6, and CXC chemokine receptor 2 (Cxcr-2). The anti-inflammatorycytokine IL-10, on the contrary, was markedly increased (FIG. 5F).Consistently, the increased expression of genes encoding tight junctionproteins (FIG. 5G-5H), thickened mucus layer (FIG. 5I), and reducedpro-inflammatory response (FIG. 5J) by C. maltaromaticum was observed inAOM-induced CRC model, confirming C. maltaromaticum could preserve thegut barrier function in CRC mice, indicating C. maltaromaticum couldreduce the pro-inflammatory response in CRC mice.

C. maltaromaticum Modulates Gut Microbiota in Apc^(min/+) Mice

To determine the effects of C. maltaromaticum inoculation on gutmicrobiota composition, 16S rRNA gene sequencing with Apc^(min/+) mousefecal samples was performed. Daily administration C. maltaromaticumcaused a distinct trend on β-diversity as revealed by PrincipalCoordinate Analysis (PCoA) (FIG. 6A). Bacteria genera ofDesulfovibrionaceae and Rikenellaceae were significantly enriched in C.maltaromaticum-gavaged mice as compared with both BHI and E. coli strainMG1655 (FIG. 6B). The abundance of some well-characterized commensalprobiotics including Bifidobacterium, butyrate producing Rosebutia, andClostridiales were significantly increased in the C.maltaromaticum-treated mice. In contrast, certain pathogenic species,including Candidatus Arthromitus, Prevotella, Coprococcus, and Dorea,exhibited significantly decreased abundance in C. maltaromaticum-gavagedmice (FIG. 6C), suggesting that the modulatory effect of C.maltaromaticum on the gut microbiota is associated with itsCRC-preventive effect.

C. maltaromaticum Increases the Abundance of Vitamin D Metabolites inMouse Models of CRC

To determine whether C. maltaromaticum could exert any effect on themetabolome, the fecal metabolites from C. maltaromaticum-treatedApc^(min/+) mice were profiled by LC-MS/MS. A significant overallcompositional alteration of the gut metabolites was observed among theC. maltaromaticum and control groups in the Apc^(min/+) mouse model(FIG. 7A). Forty-two metabolites were found to be enriched in the C.maltaromaticum-treated Apc^(min/+) mice as compared with both BHI- andE. coli strain MG1655-treated groups (FIG. 7B). Most of thesemetabolites, such as 29-norcycloartane-3,24-dione,4,4′-methylenebis(2,6-di-tert-butylphenol),3-tert-butyl-5-methylcatechol, (−)-tylocrebrine, betulinic acid andcinmyl propiote have been reported to have anti-inflammatory/anti-cancereffect. In particular, enrichment of a repertoir of vitamin Dmetabolites was observed in the fecal samples of C.maltaromaticum-treated mice. Further metabolic pathway enrichmentanalysis revealed the significant alteration of fecal vitamin D₃metabolism in C. maltaromaticum-treated Apc^(min/+) mice as comparedwith both control groups (FIG. 7C). 1,25(OH)₂D₃ is the active form ofvitamin D₃ and plays an important role in cancer prevention andtreatment¹⁴. The accumulation of considerable amounts of 1,25(OH)₂D₃ incolonocytes could reach levels that would locally induce growthinhibitory on CRC cells¹⁵. As intracellular concentrations of1,25(OH)₂D₃ could drive the biological effect of vitamin D in cellgrowth regulation, the colonic concentration of 1,25(OH)₂D₃ inApc^(min/+) mice after C. maltaromaticum treatment was further examined.As shown in FIG. 7D, C. maltaromaticum significantly increased thecolonic 1,25(OH)₂D₃ concentration. The enzymes (CYP2R1, CYP27A1, andCYP27B1) responsible for the biosynthesis of 1,25(OH)₂D₃ were alsoupregulated in the colon tissues of Apc^(min/+) mice receiving C.maltaromaticum (FIG. 7E), suggesting the potential contribution ofvitamin D metabolism in mediating the CRC-suppressive effect of C.maltaromaticum. To further validate this observation, the fecalmetabolic changes were profiled using the stool samples from AOM-inducedCRC model. Consistently, vitamin D metabolites, especially the activeform 1,25(OH)₂D₃, was found to be increased significantly in the fecalsamples of C. maltaromaticum-treated, AOM-induced CRC mice (FIG. 7F).The colonic 1,25(OH)₂D₃ level (FIG. 7G) and the enzymes responsible for1,25(OH)₂D₃ biosynthesis (FIG. 7H) were also significantly increasedafter C. maltaromaticum treatment. These results indicate that C.maltaromaticum administration increased the fecal abundance of vitaminD-related metabolites, which were activated by the colonocytes' enzymesto induce the local accumulation of 1,25(OH)₂D₃ in mouse models of CRC.

C. maltaromaticum Induces Colonic Vitamin D Receptor Activity in MouseModels of CRC

1,25 (OH)₂D₃ is synthesized and degraded in colonocytes and, when boundto its receptor, has antiproliferative activity¹⁵. To investigate thetranscriptional activation of vitamin D receptor (VDR) by C.maltaromaticum, transcriptome-sequencing was performed in colon tissuesfrom Apc^(min/+) mice under different treatments (FIG. 8A). It wasdiscovered that VDR was the only changed bile acid receptor, which wassignificantly upregulated in the C. maltaromaticum-treated Apc^(min/+)mice colon as compared with both BHI- and E. coli strain MG1655-treatedgroups (FIG. 8B). Concordantly, gene set enrichment analysis (GSEA) oftranscriptome profiles revealed that VDR signaling was significantlyenriched in the colonic tissues of C. maltaromaticum-treated Apc^(min/+)mice compared with both control groups (FIG. 8C). The upregulation ofVDR was further validated at mRNA and protein levels in C.maltaromaticum-treated Apc^(min/+) mice (FIG. 8D). The activated VDR wasthen confirmed by increased level of CRAMP (FIG. 8E), a well-known VDRtarget gene⁸, and the nuclear translocation of VDR in colonic tissues ofC. maltaromaticum-treated Apc^(min/+) mice (FIG. 8F). This finding wasfurther validated in TCGA cohort, which showed that CRC patients withhigher abundance of C. maltaromaticum exhibited higher level of VDR(FIG. 8H). Accordingly, 20 VDR target genes were upregulated in the C.maltaromaticum-treated Apc^(min/+) mice as revealed bytranscriptome-sequencing (FIG. 8G). Five of them (SLC35A4, ELF4, ELL,AGPAT1 and DENND6B) were significantly correlated with C. maltaromaticumabundance in TCGA dataset (FIG. 8H), indicating that C. maltaromaticumincreases the colonic 1,25(OH)₂D₃ level to induce the activation of VDRin Apc^(min/+) mice. The activation of VDR was further validated in theAOM-induced CRC model. Consistent results were observed as in theApc^(min/+) mice that C. maltaromaticum administration increased VDR(FIG. 9A) and its target gene (CRAMP) (FIG. 9B) expression in colonictissues and induced VDR translocation into the nucleus (FIG. 9C). Takentogether, these data indicate that the tumor-suppressive effect of C.maltaromaticum is associated with increased vitamin D metabolites andthe activation of VDR signaling.

Discussion

In the present study, the present inventors confirmed that C.maltaromaticum was depleted in CRC patients and was a potential markerfor predicting the survival of CRC patients. Co-incubation of C.maltaromaticum with colon cells significantly suppressed theproliferation and induced cell cycle arrest in CRC cells but not in thenormal colonic epithelial cells. Oral administration of C.maltaromaticum retarded the intestinal tumorigenesis in two murine CRCanimal models, supporting the anti-CRC efficacy of C. maltaromaticum.

Dysfunction of mucosal barrier function has been a common feature ofCRC. A leaky gut results in uncontrolled translocation of commensalpathogens and antigens into the body¹⁶. In this study, C. maltaromaticumwas found to restore gut barrier function through increasing theexpression of a number of gut barrier-associated markers, includingZO-1, Occludin, and E-cadherin in mouse CRC models; enhancing thethickness of the colonic inner mucus layer (a physical barrier), alongwith decreasing paracellular gap and serum LPS level in C.maltaromaticum-treated mice. A healthy barrier function is the premiseof gut homeostasis¹⁷. Any disruption or even slight break of the barriercan trigger inflammation³. Consistently, the pro-inflammatory genes ofIL-1β, IL-6, and Cxcr-2 were significantly decreased, while theanti-inflammatory cytokine IL-10 increased by C. maltaromaticumtreatment in both mouse models. The reduced expression ofpro-inflammatory cytokines (Spp1, Ccl4, Cxcl9, and Ccl5) and thedownregulated NF-kappa B signaling, which have been implicated ininflammation and carcinogenesis¹⁸, were also observed in the C.maltaromaticum treatment group in the RNA sequencing data (FIG. 10 ),indicating the involvement of C. maltaromaticum in suppressing thepro-inflammatory signaling pathway. It is therefore biologicallyplausible that the tumor-suppressive effect of C. maltaromaticum isassociated with preserved gut barrier function and reduced inflammation.

Gut microbiota dysbiosis contributes to the development of CRC^(1,19).The microbes profiling revealed that administration of C. maltaromaticumsignificantly enriched the abundance of well-characterized probiotics,such as Desulfovibrionaceae, which is known to reduce hydrogen sulphideproduction²⁰. The Rikenellaceae, which is inversely associated withpro-inflammatory cytokines and positively correlated with gut barrierprotein expression²¹, also increased at the genus level. Also, theBifidobacterium, butyrate-producing Rosebutia, Ruminococcaceae andClostridiales were increased in abundance in C. maltaromaticum-gavagedApc^(min/+) mice. On the other hand, some pathogenic species, includingCandidatus Arthromitus²² , Prevotella ²³ , Coprococcus ²⁴, and Dorea ²⁵,which could trigger excessive inflammation and promote cancerprogression, were significantly depleted after C. maltaromaticumtreatment. Thus, C. maltaromaticum protects against CRC at least in partthrough restoring gut microbiota homeostasis by enhancing thecolonization of probiotics and depleting the potential CRC-associatedpathogens.

The effect of C. maltaromaticum in modulating gut metabolomics wasfurther examined through metabolomic profiling of mouse fecal samples.It was discovered that inoculation of C. maltaromaticum to both murinemodels increased the level of a repertoire of vitamin D metabolites.Vitamin D supplementation has been viewed as a potential strategy forCRC prevention²⁶. Vitamin D deficiency is associated with increasedrisks for CRC²⁷. Moreover, the enzymes responsible for extrarenalproduction of 1,25(OH)₂D₃, including CYP2R1, CYP27A1 and CYP27B1 wereinduced by C. maltaromaticum. Colonic 1,25 (OH)₂D₃, which is responsiblefor most of the biological actions of vitamin D, was increasedsignificantly after C. maltaromaticum consumption, presenting thepossibility that C. maltaromaticum inhibits intestinal tumorigenesis viaincreasing the mucosal concentration of 1,25(OH)₂D₃. Indeed,polymorphism of CYP27B1, the enzyme for the biosynthesis of 1,25(OH)₂D₃, dictates the risk of CRC²⁸.

VDR is a transcription factor, which is involved in a wide variety ofbiological processes, including regulation of cell proliferation anddifferentiation in normal tissue and apoptosis in tumor cells²⁹. In CRC,the VDR has been reported to inhibits tumor initiation and promotion viathe regulation of p21, p27, and E-cadherin³⁰. 1,25(OH)₂D₃, the activeform of vitamin D₃, mediates cellular functions via VDR³¹. Concordantly,it was discovered that the VDR signaling was upregulated significantlyin Apc^(min/+) mice after treatment with C. maltaromaticum. Thisobservation therefore supports the notion that C. maltaromaticumelevates the level of 1,25 (OH)₂D₃ in colon tissues, thereby activatingVDR receptor to induce the downstream anti-CRC signaling.

In summary, this study uncovers the anti-CRC effect of C.maltaromaticum. The intestinal tumor-suppressive role of C.maltaromaticum is associated with the modulated gut microbiota andmetabolites together with the preserved gut barrier function and reducedmucosal inflammation. Especially, such action is associated with theinduction of vitamin D-related metabolites and the activation of VDR.Taken together, C. maltaromaticum can be used as a novel probiotic-basedprophylactics for CRC prevention.

Example II: Lactobacillus gallinarum Modulates the Gut Microbiota andProduces Anti-Cancer Metabolites to Protec Against ColorectalTumorigenesis Background of the Invention

Colorectal cancer (CRC) is the third most commonly diagnosed malignancyand the second leading cause of cancer death in the world³². There aremany risk factors associated with CRC carcinogenesis including geneticalterations, lifestyle and environmental factors³³. Over the lastdecade, gut microbiota has been shown to play a key role in CRCdevelopment. Certain probiotic bacteria such as Streptococcusthermophilus and Lactobacillus rhamnosus have anti-carcinogenicproperties^(34,35).

Most Lactobacillus species are classified as lactic acid bacteria (LAB).LAB are generally found in fermented food, such as decomposing plantsand milk products, and they are widely accepted to be used as probioticsfor humans³⁶. The beneficial effects of LAB for diseases have beenreported extensively^(37,38), and preclinical studies have shown itsabilities to reduce chronic inflammation associated with cancerdevelopment^(39,40). Using shotgun metagenomic sequencing, the presentinventors identified a probiotic species Lactobacillus gallinarum beingsignificantly depleted in the stool of CRC patients¹³, suggesting thatit might play a role in suppressing CRC. In this study, L. gallinarumwas shown to abrogate colorectal tumorigenesis in mouse models, humanCRC-derived organoids and CRC cell lines through promoting apoptosis.This tumor-suppressing effect was attributed to indole-3-lactic acid, ametabolite produced by L. gallinarum.

Materials and Methods Animal Experiments

Male Apc^(Min/+) C57B/6 transgenic mice was used as a mouse model ofspontaneous CRC⁴¹. Apc^(Min/+) mice at 5 to 6 weeks old were dividedinto 3 groups with or without CH-223191 treatment: (1) control; (2) E.coli MG1655; and (3) L. gallinarum. L. gallinarum and E. coli MG1655were cultured in MRS broth (Difco Laboratories, Detroit, Minn.) and BHIbroth, respectively. After one day, 1.0×10⁸ colony-forming units (CFUs)bacteria were collected and resuspended in 100 ul PBS. Mice were gavagedonce daily for 8 weeks and body weight and stool were examined weekly.

For the azoxymethane (AOM)/dextran sulfate sodium (DSS) model, maleC57BL/6 mice at 6 weeks old were intraperitoneally injected with asingle dose of 10 mg/kg AOM (Merck, Darmstadt, Germany), followed by 2%DSS (MP Biomedicals, Solon, Ohio) administration for 1 week.AOM/DSS-induced CRC mice were gavaged with the above L. gallinarium andE. coli MG1655 suspension following the same schedule.

Mouse colonoscopy (Karl Storz Endoskope, Tuttlingen, Germany) wasperformed prior to sacrifice. The colonoscope was inserted into the anusand advanced proximally under direct visualization, facilitated by airinsufflation, and representative pictures of the colon tumor from eachgroup were recorded. After 8 weeks of gavage, mice were anaesthetizedand sacrificed. Small intestines and colons of mice were longitudinallyopened and rinsed with PBS. Total number of tumors in small intestineand colon were recorded. Size of each tumor was measured using previouspublished formula⁴². All animal studies were performed in accordancewith guidelines approved by the Animal Experimentation Ethics Committeeof The Chinese University of Hong Kong.

DNA Extraction, 16S Ribosomal DNA Gene Amplification, from ApcMin/+Mouse Stool Samples after Gavage of L. Gallinarium

Apc^(Min/+) mouse stool samples were disrupted by bead-beating afterdigesting with mutanolysin (10 U/ul, Sigma-Aldrich) and lysozyme enzymecocktail, as described in our previous study⁷. DNA extraction andpurification were performed using DNeasy PowerSoil kit (Qiagen, Hilden,Germany). Amplicon library for bidirectional (466 bp) sequencing onIllumina MiSeq platform was constructed using universal primers 341f(SEQ ID NO:1), 5′-CCTAYGGGRBGCASCAG-3′ and 806r (SEQ ID NO:2),5′-GGACTACNNGGGTATCTAAT-3′ targeting across 16S rRNA genes V3-V4hypervariable regions. Library clean-up and normalization was performedusing the NEBNext Ultra DNA Library Pre kit (New England Biolabs,Ipswich, Mass.).

16S rRNA Gene Sequence Analysis

Raw de-multiplexed FASTQ files were preprocessed in Mothur⁴³. Contigswere created using Needleman-Wunsch alignment algorithm with defaultparameters⁴⁴, and aligned against the SILVA database (version 123) usingthe NAST algorithm^(45,46). Any contigs with homopolymers of greaterthan 8 nucleotides were removed and all that mapped within the identicalcoordinates were retained. Any sequence pairs with mismatch differenceof ≤2 were preclustered to reduce amplicon sequencing noises. Chimericsequences were culled using de novo UChime⁴⁷. Post-quality controlledsequences were classified using Greengenes 16S rRNA database (13.8). Anysequences of eukaryotic, archaea, mitochondrial, chloroplastic, orunknown origins were discarded before being binned into operationaltaxonomic units (OTUs) at 97% identity threshold. The lowest taxonomicannotation for an OTU was defined as having a consensus assignment scoreof ≥80. Sequence count table was rarefied to the smallest number ofreads per sample (i.e., 44,845 reads) to reduce the effects of variablesequencing depths on downstream analyses. Differential abundanceanalysis was performed with one way analysis of variance. Average foldchange for each OTU and heatmap was computed in the R Project forStatistical Computing⁴⁸.

Bacterial Strains and Culture Conditions

L. gallinarum (ATCC 33199) was purchased from American Type CultureCollection (ATCC; Manassas, Va.). Escherichia coli (E. coli) strainMG1655 (ATCC 700926), a non-pathogenic human commensal intestinalbacterium, was included as a negative control⁴⁹ . L. gallinarum and E.coli MG1655 were cultured in MRS broth (Difco Laboratories, Detroit,Minn.) and BHI broth, respectively, at 37° C. under aerobic condition.The bacteria were centrifuged at 5,000 rpm for 10 minutes to obtain thebacteria pallet and resuspended in BHI before gavaging to mice.

Culture Supernatant of L. gallinarum

After culturing L. gallinarum or E. coli MG1655 for 1-2 days, thebacterial concentration in each culture medium was measured usingNanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, Del.).BHI cultures were diluted to ensure equal concentrations of L.gallinarum and E. coli. Culture supernatants were then collected bycentrifugation at 5,000 rpm for 10 minutes, followed by sterilefiltration with a 0.22-μm membrane. Filtrates were collected and termedas L. gallinarum culture supernatant (LGCS) or E. coli MG1655 culturesupernatant (ECCS).

Bacterial culture supernatant was separated into low-molecular-weight(LMW) and high-molecular-weight (HMW) using Amicon Ultra-15 CentrifugalFilter Units with a pore size of 3 kilodalton (kDa, Millipore, Bedford,Mass.). After centrifugation in a swinging bucket rotor at 4,000 g for30 minutes, cells were cultured in medium containing LGCS, ECCS orcontrol broth (LMW, 10%; HMW, 1%). The bacterial supernatants wereheated at 100° C. for 30 minutes or treated with proteinase k (50 ug/ml;QIAGEN GmbH, Hilden, Germany). 10% heat-inactivated or proteinasek-treated bacterial culture supernatant was used for MTT assay.

Cell Culture

Colon cancer cell lines, HCT116 and LoVo, were purchased from ATCC. Anormal colonic epithelial cell line, NCM460, was obtained from INCELLCorporation (San Antonio, Tex.) as control. All cells were grown inhigh-glucose Dulbecco's Modified Eagle's Medium (DMEM) (Thermo FisherScientific) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher Scientific), 2 mM L-glutamine, 50 U/ml penicillin and 50 μg/mlstreptomycin in a humidified atmosphere containing 5% CO₂.

CRC Patient-Derived Organoid Culture

CRC organoids derived from 2 patients (74 and 816) were obtained fromPrincess Margaret Living Biobank (Toronto, Ontario, Canada), andembedded in Matrigel (Corning Inc, Corning, N.Y.). Culture medium waschanged every 2 days. After 5 days of culture, Matrigel was removed toexpose organoids by mechanical stress and/or TrypLE digestion(Sigma-Aldrich). Organoids were then collected for further experiments.

Cell Viability Assay

Cell viability was measured by3-(4,5-dimethylthiazoly-2-yl)-2,5-diphenylte-trazolium bromide (MTT,Sigma-Aldrich) assay. Cells were seeded on 96-well plates at 5.0×10²,1.0×10³ and 3.0×10³ cells per well and incubated for 24 hours beforetreatment. Cells were cultured in DMEM with addition of bacterialsupernatants at different concentrations (5%, 10%, or 20%) or bacteriaalone (multiplicities of infections: MOIs 50, 100). For bacterialco-culture treatment, the medium was replaced with DMEM supplementedwith 10% FBS, 1% penicillin-streptomycin and 20 μgml⁻¹ gentamycin after2 hours of co-culture under aerobic conditions before MTT assay. Cellproliferation was measured by MTT assay for 5 consecutive days. Theamount of MTT formazan product was determined by measuring absorbance ata wavelength of 570 nm (OD570) with a microplate reader (Multiskan GOMicroplate Spectrophotometer, Thermo Scientific, Vantaa, Finland). Forpharmacological inhibition of aryl hydrocarbon receptor (AhR), CH-223191(100 nM) was added 12 hours before the treatment with bacteria culturesupernatant.

Colony Formation Assay

Cells were seeded overnight on 12-well microplates at 5.0×10², 1.0×10³and 3.0×10³ cells per well. 20% of LGCS, ECCS or BHI broth was thenadded to the medium and cultured for 14 to 21 days. Cells were washedwith phosphate-buffered saline (PBS) and fixed in methanol, prior tostaining with 0.5% crystal violet. Colonies were counted manually andrelative colony formation was calculated using formula: relative colonyformation (%)=(number of colony formed/average colony number in controlgroup)×100%.

Apoptosis Assay and Cell Cycle Analysis

Cells were plated on 6-well plates 24 hours prior to treatment, andcultured in medium containing 10% LGCS, ECCS or control broth. CRCpatient-derived organoids were cultured on 6-well plates, and 10% LGCS,ECCS or control broth was added to Matrigel and growth medium. Aftertreatment, cells and organoids were digested in 0.25% trypsin-EDTA(Gibco-Invitrogen Corp., Grand Island, N.Y.) and TrypLE, respectively.For apoptosis assay, the proportion of apoptotic cells was evaluated bydual staining with Annexin V-PE and 7-Aminoactinomycin D (7-AAD) (BDPharmingen, San Jose, Calif.). Combination of Annexin V-PE and 7-AADstaining distinguished early apoptotic cells (Annexin V+, 7-AAD−) andlate apoptotic cells (Annexin V+, 7-AAD+). For cell cycle analysis,cells treated with 10% LGCS, ECCS or control broth for 1 day were fixedand stained with 50 μg/ml propidium iodide (PI) (BD Pharmingen). Cellcycle of all stained cells were analyzed using FASAria cell sorter (BDBiosciences, San Jose, Calif.).

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis

Metabonomics was performed by BIOTREE, Shanghai, China, as described inExample I.

High-Throughput Targeted Metabolomics for Tryptophan

Stock solutions were individually prepared by dissolving or dilutingeach standard substance to give a final concentration of 1 mmol/L. Analiquot of each of the stock solutions was transferred to an Eppendorftube form a mixed working standard solution. A series of calibrationstandard solutions were then prepared by stepwise dilution of this mixedstandard solution (containing isotopically-labelled internal standardmixture in identical concentrations with the samples). The metabolitesform stool and LGCS-LMW were extracted and subjected to UHPLC-MS/MSanalysis (BIOTREE, Shanghai, China).

Statistical Analysis

Values are expressed as mean±standard deviation (SD) for both in vivoand in vitro experiments. Comparisons between two groups were performedusing a two-sided Student's t-test. ANOVA was used to comparedifferences among multiple groups, and post-hoc analysis was performedby Tukey's multiple comparisons test. P-values<0.05 indicate statisticalsignificances.

Results

L. gallinarum Protects Against Intestinal Tumorigenesis in Apc^(Min/+)Mice

To investigate the effect of L. gallinarum on colorectal tumorigenesis,Apc^(Min/+) mice were first used. The mice were gavaged with L.gallinarum (1.0×10⁸ CFUs per mouse), a non-tumorigenic E. coli strainMG1655 (1.0×10⁸ CFUs per mouse) as bacteria control, or plain culturebroth once daily for 8 weeks (FIG. 11A). During the period of gavage,there was no difference in body weight among groups (FIG. 11C). Theincidence of bloody stool in L. gallinarum group was lower than theBroth control group (p<0.05) after gavage for 8 weeks (FIG. 11C).Colonoscopy identified that colon tumor sizes in L. gallinarum groupwere visually smaller than the E. coli MG1655 or broth control groups(FIG. 11B). After sacrifice, significant reductions in tumor number (E.coli, p=0.034; broth control, p=0.005) and tumor size (E. coli, p<0.05;broth control, p<0.05) were observed in the colon of L.gallinarum-treated Apc^(Min/+) mice (FIG. 11 E). L. gallinarum alsosignificantly decreased tumor number (E. coli, p=0.003; broth control,p<0.05) and tumor size (E. coli, p=0.0012; broth control, p<0.05) in thesmall intestine of Apc^(Min/+) mice (FIG. 11F). These results indicatethat L. gallinarum abrogates intestinal tumorigenesis in Apc^(Min/+)mice.

L. gallinarum Protects Against Intestinal Tumorigenesis inAOM/DSS-Induced CRC Mice

To validate the tumor-suppressive effect of L. gallinarum on colorectaltumorigenesis, a colitis-associated CRC model was established, in which6-week-old C57BL/6 mice were intraperitoneally injected with 10 mg/kgAOM, followed by 2% DSS administration for 1 week (FIG. 12A). L.gallinarum significantly reduced colorectal tumor number (E. coli,p<0.05; broth control, p=0.007) and tumor size (E. coli, p=0.019; Brothcontrol, p=0.0013) (FIGS. 12B and 12C), indicating that L. gallinarumalso suppresses intestinal tumorigenesis in AOM/DSS-induced CRC mice.

L. gallinarum Modulates the Gut Microbiota of Apc^(Min/+) Mice

To investigate the effects of L. gallinarum on gut microbiota, 16S rRNAgene sequencing was performed on Apc^(Min/+) mouse stool samples aftergavage of L. gallinarum for 8 weeks. The microbial abundance in L.gallinarum group significantly increased compared with broth control(p<0.05), while there was no difference between L. gallinarum and E.coli groups (FIG. 13A). Similarly, although the β-diversity of stoolfrom L. gallinarum-gavaged mice had a distinct trend compared with othertwo groups, there was no statistical significance (FIG. 13B). However,L. gallinarum could enhance abundances of some well-characterizedcommensal probiotics including Lactobacillus helveticus, Lactobacillusreuteri and OTUs from the Bacteroidetes phylum (FIG. 13C). Moreover,some genera, such as Alistipes, Allobaculum, Dorea, Odoribacter,Parabacteroides and Ruminococcus with species of pathogenic potentials,exhibited significantly decreased abundances in mice treated with L.gallinarum compared with control groups (FIG. 13C). Taken together,although L. gallinarum did not change the overall gut microbiotacomposition, it enriched the abundance of probiotics and potentiallydeplete gut pathogens.

L. gallinarum Supernatant Inhibits the Viability of Colon Cancer Cells

To validate the tumor-suppressive effect of L. gallinarum in vivo, invitro functional analyses were performed using two CRC cell lines(HCT116 and LoVo) and a normal colonic epithelial cell line (NCM460) ascontrol. Treatment with the culture supernatant of L. gallinarumsignificantly reduced the viability of CRC cell lines in aconcentration-dependent manner, but not in normal colonic epithelialcell line as determined by cell viability assay (FIG. 14A-C). Similarresults were observed in colony formation assay in which HCT116 (ECCS,p=0.0002; control broth, p=0.0003) and LoVo (ECCS, p=0.0001; controlbroth, p=7.6×10⁻⁵) showed significant decrease in colony compared withECCS or broth control groups (FIG. 14D). These data indicate that thesecreted molecules from L. gallinarum, can suppress the viability andcolony-forming ability of CRC cells.

L. gallinarum Supernatant Promotes Apoptosis in CRC Cells

To determine the mechanism by which LGCS suppresses CRC cell viability,the effects of LGCS on apoptosis and cell cycle distribution wereassessed quantitatively by flow cytometry with Annexin V-PE and 7-AADstaining. It was discovered that LGCS significantly promoted apoptosisin CRC cell lines, HCT116 (ECCS, p=3.3×10⁻⁶; control broth, p=5.1×10⁻⁶)(FIG. 15A) and LoVo (ECCS, p=1.4×10⁻⁶; control broth, p=3.6×10⁻⁶) (FIG.15B), while it had no effect on normal epithelial colonic cells, NCM460(FIG. 15C). Moreover, this apoptosis-inducing property of LGCS wasconfirmed on CRC organoids derived from 2 patients, 74 (ECCS,p=1.2×10⁻⁵; control broth, p=0.0001) and 816 (ECCS, p=0.0013; controlbroth, p=0.0016) (FIGS. 15D and 15E). By contrast, LGCS had no effect oncell cycle distribution in CRC cells (FIG. 15F-H).

Anti-Tumor Molecules Produced from L. gallinarum are Non-Protein with aMolecular Weight<3 kDa

To investigate the features of molecules produced from L. gallinarumresponsible for anti-CRC activity, bacterial culture supernatant wasseparated into LMW (<3 kDa) and HMW (>3 kDa) fractions using 3-kDafilter units. Decrease in viability of CRC cells was observed only inthose treated with LGCS LMW fraction, while LGCS HMW fraction had nosuppressive effect on CRC cells (FIG. 16A). Meanwhile, bothheat-inactivated-LGCS (FIG. 16B) and proteinase K-treated-LGCS retainedthe ability to reduce cell the viability of CRC cells (FIG. 16C).Collectively, these results indicated that the anti-CRC properties of L.gallinarum could be induced by non-protein molecules with a molecularweight<3 kDa.

L. gallinarum Produce and Catabolize L-Tryptophan to ReleaseIndole-3-Lactic Acid to Protect Against CRC

LC-MS/MS was next performed to identify the anti-CRC metabolite(s) inthe LGCS-LMW fraction Score plots of principal component analysis (PCA)showed clear separations among the LMW fractions of LGCS, ECCS andcontrol broth groups with LMW (FIG. 17A). Differential abundanceanalysis showed the critical products generated from L. gallinarum maycontribute to the anti-CRC effects (FIG. 17B). To prove L. gallinarumproduced LMW tumor-suppressive molecules are to some extent responsiblefor the anti-CRC effect of L. gallinarum in vivo, the intestinemetabolomics were then examined by using the fecal samples fromApc^(Min/+) mice under different treatments. Daily administration of L.gallinarum caused a significant overall compositional alteration of thegut metabolites as revealed by PCoA (FIG. 17C). Among the identifiedcritical products, four metabolites including palmitic acid, 4-Pyridoxicacid, L-tryptophan (Try) and gamma-L-Glutamyl-L-glutamic acid were foundto be overlapped in both the bacteria culture supernatant and theApc^(Min/+) mice (FIG. 17D). Notably, Try was the only one metabolite,which was enriched significantly in the LGCS-LMW fraction and the L.gallinarum-treated Apc^(Min/+) mice, suggesting the possibility that Trycan be secreted from L. gallinarum directly. Try is an essential aminoacid for host, intake of Try from dietary may reach the colon, where thebacteria will degrade them into its catabolites⁵⁰. Concordantly, L.gallinarum administration was found to cause enrichment of Trycatabolites, especially the indole derivatives in Apc^(Min/+) mice (FIG.17E). It was hypothesized that L. gallinarum is the Try and/or Trycatabolites producer.

Next, further confirmation was sought for the catabolizing and/orproduction of Try form L. gallinarum through high throughput targetedTry metabolic profiling. The PCA regarding Try and its downstreammetabolites showed obvious separations among control, ECCS and LGCSgroups (FIG. 18A). Differential abundance analysis showed that Try levelwas decreased in LGCS-LMW (FIG. 18C), suggesting the Try catabolizingcapacity of L. gallinarum. The catabolizes of Try were then examined,and it was found that indole-3 lactic acid (ILA), which was one of thedownstream catabolites from Try, enriched in both the LGCS-LMW and thestool samples from L. gallinarum-treated Apc^(Min/+) mice (FIG.18B-18D). The cell viability of CRC cells decreased substantially whenexposed to the same amount of ILA as produced form L. gallinarum (FIG.18E), indicating that L. gallinarum can produce Try, and at the sametime can use Try to produce Try catabolites.

Functional investigation of ILA in CRC tumorigenesis was then performedin vivo. As shown in FIG. 18G, gavage of ILA (20 mg/kg) into Apc^(Min/+)mice significantly reduced tumor number (FIG. 18H) and tumor size (FIG.18I). The amount of cells with positive TUNEL staining was alsoincreased in ILA-treated tumor tissues, but not in adjacent normaltissues (FIG. 18J). Moreover, additional experiments were performed toinvestigate if antagonizing ILA could abrogate L. gallinarum medicatedanti-cancer effect. As shown in S FIG. 18K, blocking ILA receptor, arylhydrocarbon receptor (AhR), using AhR antagonist (CH-223191)²² ²³abolished the effect of L. gallinarum in suppressing CRC in vitro (FIG.18L) and in Apc^(Min/+) mice (FIG. 18M-18R). Collectively, these dataindicate that L. gallinarum can produce L-tryptophan, and at the sametime, convert it to its downstream ILA and other metabolites. Thus, ILAcan contribute, at least in part, to the tumor-suppressive effect of L.gallinarum.

Discussion

This study has demonstrated for the first time that oral administrationof L. gallinarum reduced intestinal tumor number and size in Apc^(Min/+)mice and confirmed in DSS/AOM induced CRC mouse model, indicating thatL. gallinarum suppresses CRC tumorigenesis. From in vitro experiments,small non-protein metabolites produced by L. gallinarum were observed tosuppress the growth of CRC cells and CRC patient-derived organoids bypromoting apoptosis.

Certain probiotics can suppress the progression of CRC in preclinicalexperiments⁵¹⁻⁵⁶. For example, both living and heat-killed Lactobacillusrhamnosus GG (LGG) have anti-CRC effect by promoting apoptosis in humanCRC cells⁵⁷. In animal model, LGG could suppress CRC development byincreasing the expression of pro-apoptotic proteins such as Bax, casp3and p53⁵⁸. Several studies have suggested that regular consumption ofprobiotics may improve the imbalanced intestinal microbiota, thusreducing the chance of chronic inflammation and production ofcarcinogenic compounds during intestinal dysbiosis⁵⁹⁻⁶¹.

In the present study, it was discovered that L. gallinarum significantlyenriched the abundance of well-characterized commensal probiotics suchas L. helveticus and L. reuteri, while some pathogenic potential speciessuch as Alistipes, Allobaculum, Dorea, Odoribacter, Parabacteroides andRuminococcus ⁶²⁻⁶⁴ were significantly depleted in mice treated with L.gallinarum. L. reuteri is known to suppress inflammation-associatedcolon carcinogenesis by producing histamine⁶⁵. Thus, L. gallinarumsuppresses CRC at least in part through enriching the abundance ofprobiotics and depleting potential CRC pathogens. Gut microbiota plays acritical role in CRC tumorigenesis. One previous study showed thattransplantation of feces from patients with CRC can promotetumorigenesis in germ-free mice and mice receiving carcinogen AOM⁶⁶.Another study demonstrated that transplantation of fecal samples fromAOM/DSS mice to germ-free mice led to increased tumor developmentcompared with those harboring fecal samples from naive healthy mice⁶⁷.These studies suggest that gut dysbiosis contribute to tumorsusceptibility and alteration of the intestinal microbiota is animportant determinant of colon tumorigenesis. Meanwhile, some studiesfound that probiotics can alter the composition of microbiota toalleviate cancer progression. For example, Lactobacillus salivarius Rencould suppress CRC tumorigenesis via modulating intestinalmicrobiota^(68,69). These findings collectively inferred that probioticslike L. gallinarum suppress CRC development through modulating gutmicrobial composition.

This study also demonstrated that the metabolites produced by L.gallinarum suppressed CRC cell growth through inducing apoptosis. Usingmetabolomic analysis, it was revealed that L. gallinarum can not onlyproduce Try, but also catabolize Try to produce Try catabolites. Recentdata suggest that Try catabolites generated by the gut microbiota areimportant contributors in maintaining intestinal homeostasis⁵⁰. It isfurther identified that ILA, one of the Try catabolites, significantlyincreased in the LGCS and L. gallinarum-treated Apc^(Min/+) mice. ILAwas identified to be a metabolite of breastmilk tryptophan, secreted byprobiotic Bifidobacterium longum to prevent intestine inflammation⁷⁰. Inaddition, production of ILA through gut microbial was reported toalleviate colitis in mice through inhibiting epithelial autophagy. CRCis influenced by the balance between microbial production ofhealth-promoting metabolites (e.g., short-chain fatty acids) andpotentially carcinogenic metabolites (e.g., secondary bile acids⁷¹.Previous studies have demonstrated the anti-carcinogenic attributes ofprobiotic metabolites, especially for those produced by LAB^(72,73). Forexample, metabolites produced by Lactobacillus plantarum exhibitedselective cytotoxicity via provoking antiproliferative activity andinducing apoptosis against malignant cancer cells⁷⁴. Ferrichrome derivedfrom L. casei has a strong tumor-suppressive effect on CRC cells byinhibiting the JNK signaling pathway⁷⁵. Thus, the anti-CRC feature of L.gallinarum could be also at least in part contribute to its releasedprotective metabolites. However, whether ILA secreted from L. gallinarumis the main metabolite for CRC suppression requires furtherinvestigation.

In conclusion, this study is the first to demonstrate the anti-CRCeffect of L. gallinarum. L. gallinarum protects against intestinaltumorigenesis. Such action is associated with the modulation of the gutmicrobial composition and the secretion of protective metabolitesincluding ILA to promote apoptosis in cancer cells. These findings mayfacilitate the development of therapeutic strategy using probiotics forchemoprevention of CRC.

Example III: Synergistic Effect of Lactobacillus gallinarum andCarnobacterium maltaromaticum in Protecting Against ColorectalTumorigenesis Background of the Invention

A single CRC-depleted probiotic has been proved to act as a novelprophylactic for CRC prevention in mice with minimal side effects. Theunderlying mechanism between different probiotic species are quitedifferent. In light of the complexity and heterogeneity of tumors,combining different probiotics to explore its synergistic effect willprobably be a powerful weapon in the battle against CRC.

Materials and Methods Animal Experiments

Male C57BL/6 mice at 6 weeks old were intraperitoneally injected with asingle dose of 10 mg/kg AOM (Merck, Darmstadt, Germany), followed by 2%DSS (MP Biomedicals, Solon, Ohio) administration for 1 week. After DSStreatment, all the mice were randomly assigned into 5 groups: (1) BHI;(2) E. coli MG1655 (1.0×10⁸ colony-forming units (CFUs)); (3) L.gallinarum (1.0×10⁸ CFUs); (4) C. maltaromaticum (1.0×10⁸ CFUs); and (5)L. gallinarum (0.5×10⁸ CFUs)+C. maltaromaticum (0.5×10⁸ CFUs). Mice weregavaged once daily for 20 weeks. Mouse colonoscopy (Karl StorzEndoskope, Tuttlingen, Germany) was performed prior to sacrifice. Aftersacrifice, the colons of mice were longitudinally opened and rinsed withPBS. Total number of tumors in colon were recorded. All the procedureswere performed in accordance with guidelines approved by the AnimalExperimentation Ethics Committee of The Chinese University of Hong Kong.

Bacterial Strains and Culture Conditions

All the bacteria stain and culture condition were the same as used inExamples I and II.

Multicolour Flow Cytometry Analysis

Multicolour flow cytometry was performed as we described previously⁸. Inbrief, colon tissues were dissected into small pieces and digested withHank's balanced salt solution with 0.1 mg/ml collagenase D and 50 U/mlDNase I for 30 min at 37° C. on a shaking platform. The cell suspensionwas filtered through a 70-μm cell strainer and centrifuged at 500 g for20 min. The cell pallet was resuspended in Hank's media plus 1% fetalcalf serum (FCS) for cell surface marker analysis.

Statistical Analysis

Results are expressed as mean±standard deviation (SD). ANOVA was used tocompare differences among multiple groups, and post-hoc analysis wasperformed by Tukey's multiple comparisons test. P-values<0.05 indicatestatistical significances.

Combined L. gallinarum and C. maltaromaticum Synergistically ProtectsAgainst Intestinal Tumorigenesis in AOM/DSS-Induced CRC Mice

To investigate the synergistic effect of L. gallinarum and C.maltaromaticum on colorectal tumorigenesis, DSS-promoted, AOM-inducedCRC mice were randomized treated with L. gallinarum, C. maltaromaticumalone, and combination of L. gallinarum+C. maltaromaticum. BHI and anon-tumorigenic E. coli strain MG1655 were used as plain culture brothand bacteria control, respectively (FIG. 19A). After 20-week gavage,colonoscopy identified that colon tumor sizes in probiotics treatedgroup were visually smaller than the E. coli MG1655 or broth controlgroups (FIG. 19B). In accordance with this observation, the alleviationof colonic inflammation was observed in the probiotic treated group asreflected by increased colon length. Administration of the probioticssignificantly reduced the tumor number and tumor size in the colonAOM/DSS-induced CRC mice (FIG. 19C). Most strikingly, combined L.gallinarum/C. maltaromaticum led to an obvious reduction of colon tumornumber and tumor size as compared with L. gallinarum or C.maltaromaticum alone treatment, indicating combined L. gallinarum/C.maltaromaticum synergistically abrogates intestinal tumorigenesis inAOM/DSS-induced CRC mice.

Combined L. gallinarum and C. maltaromaticum Synergistically Modifiedthe Tumor Immune Microenvironment in AOM/DSS-Induced CRC Mice

To directly address the effect of probiotics on the tumor immunemicroenvironment in CRC, the composition of tumor-infiltrating immunecells isolated from the colon of AOM/DSS-induced CRC mice wasinvestigated under different treatments. Enrichment of B cells,tumor-infiltrating CD8⁺ cytotoxic T cells and depletion ofmyeloid-derived suppressor cells (MDSCs) was observed after L.gallinarum alone treatment as compared with BHI- or E. coli-treatedmice. Whereby, the nature killer (NK) cells, which could act as a“tumor-killer”, accumulated in C. maltaromaticum-treated mice, did notinfiltrate the L. gallinarum-treated mice. In addition, C.maltaromaticum alone treatment decreased the MDSCs. Of note, combined L.gallinarum/C. maltaromaticum led to a generalized anti-cancer immunemodulatory effect as revealed by upregulation of B cells, NK cells, CD8⁺cytotoxic T cells and downregulation of MDSCs (FIG. 20 ). Collectively,the results indicate that combined L. gallinarum/C. maltaromaticumaffects numerous types of immune cells, which will mediate, at least inpart of its synergistic tumor-suppressive effect.

Discussion

In this study, it was demonstrated for the first time that oraladministration of a combined L. gallinarum/C. maltaromaticum probioticscocktail suppresses tumor formation in the AOM/DSS mouse model in asynergistic manner. a significant reduction of tumor number and tumorsize was observed in the cocktail group as compared with the probioticalone treatment group. Furthermore, numerous immune cells, including Bcells, cytotoxic T cells, NK cells and MDSCs, were identified as beinginvolved in the cocktail-treated mice, which will partially mediate thetumor-suppressive effect of the probiotic cocktail. These resultsstrongly recommend combined L. gallinarum/C. maltaromaticum probioticscocktail as a therapeutic strategy for the prevention and treatment ofCRC.

Example IV: Lactococcus lactis Inhibits Colorectal Tumorigenesis ThroughSecreting Aminopeptidase Background of the Invention

Colorectal cancer (CRC) is the third most common cancer and constitutesa major health burden worldwide. Developing new prevention strategieswith minimal toxicity are highly warranted. The probiotics, whichinteracting directly with the colon epithelial cells, supported thepivotal role for preventing CRC development by using CRC-depletedprobiotics. Using shotgun metagenomic sequencing, Lactococcus lactis (L.lactis) was identified to be depleted in the stool of CRC patients ascompared with healthy subjects, suggesting that the potential protectiverole in CRC.

In this study, L. lactis was shown to inhibit colorectal tumorigenesisin Apc^(min/+) mouse model, human CRC-derived organoids, and CRC celllines. The tumor-suppressive effect is attributed to the secretion ofaminopeptidase by L. lactis.

Materials and Methods Animal Experiments

5-week-old male C57BL/6J-Apc^(min)/J mice were purchased from theJackson Laboratory (Bar Harbor, Me., USA) and maintained in the animalfacility at the Chinese University of Hong Kong. 28 mice were dividedinto 3 groups randomly. 1.0×10⁸ colony-forming units (CFUs) L. lactis,E. coli MG1655 or the same volume of BHI was administrated to them dailyfor 12 weeks. Mouse colonoscopy (Karl Storz Endoskope, Tuttlingen,Germany) was performed prior to sacrifice. Total number of tumors insmall intestine and colon were recorded after sacrifice. All animalstudies were performed in accordance with guidelines approved by theAnimal Experimentation Ethics Committee of The Chinese University ofHong Kong.

Bacterial Strains Isolation and Culture Conditions

Healthy human fecal samples were collected and suspended in the PBSwithin 30 minutes. The obtained suspension was homogenized in beater andfiltered through a 100 μm strainer to remove larger particles. The fecalsuspension was then plated on the M17 agar plate and incubated inaerobic incubator at 37° C. for 24 hours. All colonies were purified bystreak plate method and individually cultured for 24 hours in M17 broth(Thermo Fisher Scientific, West Palm Beach, Fla.) at 37° C. in aerobicincubator and the 16S rDNA for each isolate was amplified by PCR withthe university primers 341F (SEQ ID NO:1:5′-CCTAYGGGRBGCASCAG-3′) and806R (SEQ ID NO:2: 5′-GGACTACNNGGGTATCTAAT-3′). The PCR product was sentfor DNA sequence. L. lactis was identified through blasting the 16S rDNAin the GenBank.

L. lactis and E. coli MG1655 were cultured in BHI broth (Thermo FisherScientific, West Palm Beach, Fla.) at 37° C. under aerobic condition.The bacteria were centrifuged at 5,000 rpm for 10 minutes to obtain thebacteria pallet and resuspended in BHI before gavaging to mice.

Culture Supernatant of L. lactis

The density of L. lactis or E. coli MG1655 was measured using NanoDropspectrophotometer (NanoDrop Technologies, Wilmington, Del.). When theoptical density (OD)₆₀₀ reached 0.5, the bacteria culture supernatantswere centrifuged (5,000 rpm, 10 minutes) and filtered with a 0.22-μmmembrane filter. Filtrates were collected and termed as L. lactisconditioned-medium (LL.CM) or E. coli MG1655 conditioned-medium (ECCM).The bacterial conditioned-medium were heated at 100° C. for 30 minutesor treated with proteinase k (PK, 50 ug/ml; QIAGEN GmbH, Hilden,Germany) to evaluate the characteristics of the tumor-suppressivemolecule(s) secreted from L. lactis.

Cell Culture

Colon cancer cell lines, HCT116 and HT29, were purchased from ATCC. Anormal colonic epithelial cell line, NCM460, was obtained from INCELLCorporation (San Antonio, Tex.) as control. All cells were grown inhigh-glucose Dulbecco's Modified Eagle's Medium (DMEM) (Thermo FisherScientific) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher Scientific), 2 mM L-glutamine, 50 U/ml penicillin and 50 μg/mlstreptomycin in a humidified atmosphere containing 5% CO₂.

CRC Patient-Derived Organoid Culture

CRC organoids derived from 2 patients (74 and 828) were obtained fromPrincess Margaret Living Biobank (Toronto, Ontario, Canada), andembedded in Matrigel (Corning Inc, Corning, N.Y.). 5% of the bacteriaconditioned medium was used to treat the organoids. Culture medium waschanged every 2 days. Organoids' sizes were calculated by using Image J.

Cell Viability Assay

Cell viability was measured by3-(4,5-dimethylthiazoly-2-yl)-2,5-diphenylte-trazolium bromide (MTT,Sigma-Aldrich) assay. 1,000 cells were seeded on 96-well plates andtreated with bacteria conditioned-medium (5%). The amount of MTTformazan product was determined by measuring absorbance at a wavelengthof 570 nm (OD570) with a microplate reader (Multiskan GO MicroplateSpectrophotometer, Thermo Scientific, Vantaa, Finland).

Colony Formation Assay

500 cells were seeded overnight on 12-well microplates and treated withbacteria conditioned-medium (5%) for 2 weeks. Cells were washed withphosphate-buffered saline (PBS) and fixed in methanol, prior to stainingwith 0.5% crystal violet. Colonies were counted manually.

Silver Staining and Protein Identification

The anti-tumor molecule(s) were resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis (5%) (SDS-PAGE). Silverstaining was performed following the instructions provided by themanufacturer (Thermo Scientific, Rockford, USA). After staining,specific bands from the >100-kDa fraction were excised by pipette tipsinto 1.5 ml plastic tubes for in-gel digestion following themanufacturer's instructions (Thermo Scientific, Rockford, USA). Theobtained peptide mixtures were subject to mass spectrometry (MS)analysis. MS spectral data were processed using the Bruker Compass DataAnalysis software, and the generated peak lists were converted into theMascot search engine against the Swiss-Prot 51.6 database.Aminopeptidase was identified as the potential functional protein.

Statistical Analysis

Results are expressed as mean±standard deviation (SD). ANOVA was used tocompare differences among multiple groups, and post-hoc analysis wasperformed by Tukey's multiple comparisons test. P-values<0.05 indicatestatistical significances.

L. lactis Protects Against Intestinal Tumorigenesis in Apc^(Min/+) Mice

To investigate the potential protective effect of L. lactis oncolorectal tumorigenesis 5-week-old Apc^(Min/+) mice were orally gavagedwith L. lactis (1.0×10⁸ CFUs per mouse) or E. coli strain MG1655(1.0×10⁸ CFUs per mouse) for 12 weeks. BHI was used as the plain culturebroth control (FIG. 21A). Colonoscopy was performed during the period ofgavage to monitor the tumor formation (FIG. 21B, upper). The tumorhistology was further examined (FIG. 21B, lower) and the obviousreduction of total tumor number and tumor size was observed in L.lactis-treated Apc^(Min/+) mice (FIG. 21C). These results indicate thatL. lactis abrogates intestinal tumorigenesis in Apc^(Min/+) mice.

L. lactis Conditioned Medium (LL.CM) Inhibits the Viability of ColonCancer Cells

To determine the tumor-suppressive effect of L. lactis in vitro, coloncancer cell lines (HCT116 and HT29) and a normal colonic epithelial cellline (NCM460) were co-cultured with or without LL.CM. As shown in FIG.22A, co-culture with LL.CM significantly reduced the viability of CRCcell lines, but not in normal colonic epithelial cell line as determinedby MTT assay. This inhibitory effect of LL.CM was further confirmed onCRC patient derived organoids. Consistent results were observed thatLL.CM suppresses the organoids' size significantly as compared with BHIand Ec.CM (FIG. 22B). These data indicate that the secreted moleculesfrom L. lactis, can suppress the viability of CRC cells in vitro.

Anti-Tumor Molecules Produced from L. lactis are Protein(s) with aMolecular Weight>100 kDa

To determine the characteristic of molecules produced from L. lactisresponsible for anti-CRC activity, the LL.CM was inactivated by heatingat 100° C. for 30 minutes, the inhibitory effect of LL.CM disappeared(FIG. 23A, upper). Consistently, digestion LL.CM by protease K (PK, 50μg/mL) further neutralized the tumor-suppressive effect of LL.CM (FIG.23A, lower), indicating the protein nature of the anti-tumor molecule(s)in the LL.CM. The colony formation was further performed to confirm theanti-tumor fraction separated from the LL.CM was protein (FIG. 23B).LL.CM was further separated into LMW 100 kDa) and HMW (>100 kDa)fractions using 100-kDa filter units. As shown in FIG. 23C, decrease inviability of CRC cells was observed only in those treated with LL.CM HMWfraction, while LL.CM LMW fraction had no suppressive effect on CRCcells. Collectively, these results indicate that the anti-CRC propertiesof L. lactis could be induced by one or more proteins with a molecularweight>100 kDa.

Anti-Tumor Molecules Produced from L. lactis Contains Aminopeptidase

The potential functional secreted proteins were separated by 5% SDS-PAGEand in-gel digestion, which was then analyzed by mass spectrometry. Theresults show that aminopeptidase was enriched in the LL.CM>100 kDafaction (FIG. 24 ). These results indicate that the anti-tumor fractionsecreted from L. lactis might be an aminopeptidase.

Discussion

In this study, oral administration of L. lactis was demonstrated toreduce intestinal tumor number and size in Apc^(Min/+) mice. Further invitro experiments revealed that a protein with molecular weight>100 kDaattributes to the tumor-suppressive effect, and this protein might beaminopeptidase. This healthy human isolated probiotic could be used as apotential prophylactic for preventing CRC in human with minimal sideeffects.

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All patents, patent applications, and other publications, includingGenBank Accession Numbers and equivalents, cited in this application areincorporated by reference in the entirety for all purposes.

1. A composition for use in treating or preventing colorectal cancer (CRC) in a subject comprising an effective amount of (1) Lactobacillus gallinarum, indole-3-lactic acid (ILA), Lactococcus lactis, or an aminopeptidase produced by L. lactis having a molecular weight greater than 100 kDa; and (2) a physiologically acceptable excipient.
 2. The composition of claim 1, further comprising an effective amount of Carnobacterium maltaromaticum.
 3. The composition of claim 1, comprising an effective amount of combination of (a) L. gallinarum or L. lactis and (b) C. maltaromaticum.
 4. The composition of claim 1, which is formulated for oral ingestion.
 5. The composition of claim 4, which is in the form of a food or beverage item, or a food or beverage additive.
 6. The composition of claim 1, which is in the form of a powder, liquid, paste, cream, tablet, capsule, or caplet.
 7. The composition of claim 1, wherein L. gallinarum or L. lactis is present in the range of about 1×10⁸ to about 1×10¹² CFU per gram weight of the composition.
 8. The composition of claim 2, wherein C. maltaromaticum is present in the range of about 1×10⁸ to about 1×10¹² CFU per gram weight of the composition.
 9. The composition of claim 2, wherein the CFU ratio of L. gallinarum or L. lactis to C. maltaromaticum ranges from about 1:5 to about 5:1.
 10. The composition of claim 9, which is formulated in a daily dosage.
 11. A method for treating or reducing risk of colorectal cancer in a subject, comprising administering to the subject the composition of claim
 1. 12. The method of claim 11, wherein the composition comprises an effective amount of (i) Lactobacillus gallinarum or Lactococcus lactis (ii) L. gallinarum or L. lactis culture supernatant; (iii) ILA, or (iv) an aminopeptidase produced by L. lactis with a molecular weight of greater than 100 kDa.
 13. The method of claim 11, wherein the composition further comprises an effective amount of Carnobacterium maltaromaticum.
 14. The method of claim 11, wherein the administering step comprises administering to the subject a first composition comprising an effective amount of (i) L. gallinarum or L. lactis; (ii) L. gallinarum or L. lactis culture supernatant; (iii) ILA; or (iv) an aminopeptidase produced by L. lactis with a molecular weight of greater than 100 kDa, and administering to the subject a second composition comprising an effective amount of C. maltaromaticum.
 15. The method of claim 13, wherein the CFU ratio of L. gallinarum or L. lactis to C. maltaromaticum ranges from about 1:5 to about 5:1.
 16. The method of claim 11, wherein the administering step comprises oral ingestion of the composition(s).
 17. The method of claim 11, wherein the subject is not diagnosed with colitis.
 18. A kit for treating or preventing colorectal cancer in a subject comprising (1) a first container containing a first composition comprising an effective amount of L. gallinarum, L. lactis, ILA, or an aminopeptidase produced by L. lactis with a molecular weight of greater than 100 kDa, and (2) a second container containing a second composition comprising an effective amount of C. maltaromaticum.
 19. The kit of claim 18, wherein L. gallinarum or L. lactis is present in the first composition in the range of about 1×10⁸ to about 1×10¹² CFU per gram weight of the first composition, and C. maltaromaticum is present in the second composition in the range of about 1×10⁸ to about 1×10¹² CFU per gram weight of the second composition.
 20. The kit of claim 18, wherein the compositions are in the form of a powder, liquid, paste, cream, tablet, capsule, or caplet. 